Antigen binding protein and its use as addressing product for the treatment of cancer

ABSTRACT

The present invention relates to a novel antigen binding protein, in particular a monoclonal antibody, capable of binding specifically to the protein Axl as well as the amino and nucleic acid sequences coding for said protein. From one aspect, the invention relates to a novel antigen binding protein, or antigen binding fragments, capable of binding specifically to Axl and, by inducing internalization of Axl, being internalized into the cell. The invention also comprises the use of said antigen binding protein as an addressing product in conjugation with other anti-cancer compounds, such as toxins, radio-elements or drugs, and the use of same for the treatment of certain cancers.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.13/935,918, filed Jul. 5, 2013, now issued U.S. Pat. No. 9,173,962,which is a continuation-in-part of International Application No.PCT/EP2012/071832, filed Nov. 5, 2012, and claims the priority ofEuropean Patent Application No. EP11306416.6, filed Nov. 3, 2011, thecontent of all of which is incorporated herein by reference.

The present invention relates to a novel antigen binding protein, inparticular a monoclonal antibody, capable of binding specifically to theprotein Axl as well as the amino and nucleic acid sequences coding forsaid protein. From one aspect, the invention relates to a novel antigenbinding protein, or antigen binding fragments, capable of bindingspecifically to Axl and, by inducing internalization of Axl, beinginternalized into the cell. The invention also comprises the use of saidantigen binding protein as an addressing product in conjugation withother anti-cancer compounds, such as toxins, radio-elements or drugs,and the use of same for the treatment of certain cancers. The inventionrelates more particularly to a novel antibody-drug conjugate for thetreatment of cancer.

“Axl” (also referred to as “Ufo”, “Ark” or “Tyro7”) was cloned frompatients with chronic myeloid leukemia as an oncogene triggering thetransformation when over-expressed by mouse NIH3T3. It belongs to afamily of receptor tyrosine kinases (RTKs) called the TAM (Tyro3, Axl,Mer) family, which includes Tyro3 (Rse, Sky, Dtk, Etk, Brt, Tif), Axl,and Mer (Eyk, Nyk, Tyro-12) [Lemke G. Nat. Rev. Immunol. (2008).8,327-336].

The human protein Axl is a 894 amino acids protein which sequence isrepresented in the sequence listing as SEQ ID NO. 29. Amino acids 1-25corresponding to the signal peptide, the human protein Axl, without thesaid peptide signal, is represented in the sequence listing as SEQ IDNO. 30.

Gas6, originally isolated as growth arrest-specific gene, is the commonligand for the members of the TAM family [Varnum B. C. et al. Nature(1995).373, 623-626]. Gas6 exhibits the highest affinity for Axl,followed by Tyro3 and finally by Mer [Nagata K. et al. J. Biol. Chem.(1996).271, 30022-30027]. Gas6 consists in a γ-carboxyglutamate(Gla)-rich domain that mediates binding to phospholipid membranes, fourepidermal growth factor-like domains, and two laminin G-like (LG)domains [Manfioletti G., Brancolini, C., Avanzi, G. & Schneider, C. Mol.Cell Biol. (1993).13, 4976-4985]. As many other RTKs, ligand bindingresults in receptor dimerization and autophosphorylation of tyrosineresidues (tyrosine residues 779, 821 and 866 for the receptor Axl) whichserve as docking sites for a variety of intracellular signalingmolecules [Linger R. M. Adv. Cancer Res. (2008).100, 35-83]. Moreover,the Axl receptor can be activated through a ligand-independent process.This activation can occur when the Axl receptor is overexpressed.

Gas6/Axl signaling has been shown to regulate various cellular processesincluding cell proliferation, adhesion, migration and survival in alarge variety of cells in vitro [Hafizi S. & Dahlback, B. FEBS J.(2006).273, 5231-5244]. In addition, the TAM receptors are involved inthe control of innate immunity; they inhibit the inflammatory responsesto pathogens in dendritic cells (DCs) and macrophages. They also drivephagocytosis of apoptotic cells by these immune cells and they arerequired for the maturation and killing activity of natural killer (NK)cells [Lemke G. Nat. Rev. Immunol. (2008).8, 327-336].

Weakly expressed on normal cells, it is predominantly observed infibroblasts, myeloid progenitor cells, macrophages, neural tissues,cardiac and skeletal muscle where it supports mainly cell survival. TheGas6/Axl system plays an important role in vascular biology byregulating vascular smooth muscle cell homeostasis [Korshunov V. A.,Mohan, A. M., Georger, M. A. & Berk, B. C. Circ. Res. (2006).98,1446-1452; Korshunov V. A., Daul, M., Massett, M. P. & Berk, B. C.Hypertension (2007).50, 1057-1062].

In tumor cells, Axl plays an important role in regulating cellularinvasion and migration. Over-expression of Axl is associated not onlywith poor prognosis but also with increased invasiveness of varioushuman cancers as reported for breast, colon, esophageal carcinoma,hepatocellular, gastric, glioma, lung, melanoma, osteosarcoma, ovarian,prostate, rhabdomyosarcoma, renal, thyroid and uterine endometrialcancer [Linger R. M. Adv. Cancer Res. (2008).100, 35-83 and Verma A.Mol. Cancer Ther. (2011).10, 1763-1773, for reviews]. In breast cancer,Axl appears to be a strong effector of the Epithelial-to-mesenchymaltransition (EMT); EMT program contributes actively to migration anddissemination of cancer cells in the organism [Thiery J. P. Curr. Opin.Cell Biol. (2003).15, 740-746].

Axl has also been shown to regulate angiogenesis. Indeed knockdown ofAxl in endothelial cells impaired tube formation and migration [HollandS. J. et al. Cancer Res. (2005).65, 9294-9303] as well as disturbedspecific angiogenic signaling pathways [Li Y. et al. Oncogene (2009).28,3442-3455].

More recently several studies on a range of cellular models describedthe involvement of an Axl overexpression in drug resistance phenomena.The following table 1 summarized these studies.

TABLE 1 Reference Cancer type Therapeutic agent Cellular model Macleodet al., Ovarian Cisplatin PE01/PE01CDDP 2005 cancer Mahadevan et al.,GIST Imatinib GIST882 >GIST-R 2007 inhibitor of c- kit/PDGFR Lay et al.,2007 NSCLC Doxorubicin CL-1 clones CL1-5F4 clones Hong et al., 2008 AMLDoxorubicin/ U937 Cisplatin Liu et al., 2009 Breast Lapatinib HER2 (+)BT474 Cancer (HER1 and HER2 (J4) inhibitor) Keating et al., AstrocytomaTemozolomide G12 2010 Carboplatin A172 Vincristin Ye et al., 2010 NSCLCErlotinib HCC827

Complete references cited in table 1 above are as follow:

Macleod, K. et al. Cancer Res. (2005).65, 6789-6800

Mahadevan D. et al. Oncogene (2007).26, 3909-3919

Lay J. D. et al. Cancer Res. (2007).67, 3878-3887

Hong C. C. et al. Cancer Lett. (2008).268, 314-324

Liu L. et al. Cancer Res. (2009).69, 6871-6878

Keating A. K. et al. Mol. Cancer Ther. (2010).9, 1298-1307

Ye X. et al. Oncogene (2010).29, 5254-5264

In such a context the Axl RTK is considered as an interesting target inoncology. Several groups already developed anti-tumoral strategiestargeting the gas6/Axl axis, either using naked monoclonal antibodies ortargeted small molecules [Verma A. Mol. Cancer Ther. (2011).10,1763-1773].

In a first embodiment, the invention relates to an antigen bindingprotein, or an antigen binding fragment thereof, which i) specificallybinds to the human protein Axl, and ii) is internalized following itsbinding to said human protein Axl.

More generally, the invention relates to the use of the protein Axl forthe selection of an antigen binding protein, or an antigen bindingfragment thereof, capable of being internalized following its binding tothe said target Axl. More particularly, the said target is theextracellular domain of Axl.

In this particular aspect, the present invention is thus directed to anin vitro method for the screening of a compound, or a binding fragmentthereof, capable of delivering or internalizing a molecule of interestinto mammalian cells, said molecule of interest being covalently linkedto said compound, wherein said method comprises the following steps of:

a) selecting a compound which is capable of specifically binding the Axlprotein, or the extracellular domain (ECD) thereof, or an epitopethereof;

b) optionally, covalently linking said molecule of interest, or acontrol molecule, to said compound selected in step a) to form acomplex;

c) contacting said compound selected in step a), or said complexobtained in step b), with a mammal cell, preferably viable cell,expressing at its surface the Axl protein, or a functional fragmentthereof;

d) determining whether said compound, or said molecule of interest orsaid complex, has been intracellularly delivered or internalized intosaid mammalian cell; and

e) selecting said compound as a compound capable of delivering orinternalizing a molecule of interest into a viable mammalian cell.

In a preferred embodiment, said compound capable of delivering orinternalizing a molecule of interest into a viable mammalian cell is aprotein (also designated herein polypeptide or peptide) or aprotein-like compound comprising a peptidic structure, particularly anamino-acid sequence of at least 5, 10, 15 or more amino acids residues,said amino-acid residue(s) can be glycosylated.

When said compound capable of delivering or internalizing a molecule ofinterest into a viable mammalian cell is a protein or a protein-likecompound, said compound is also called herein an “antigen bindingprotein”, said antigen binding protein, or binding fragment thereof,can:

-   i) specifically binds to the protein Axl, preferably the human Axl    protein, and-   ii) is internalized into a mammalian cell following its binding to    said protein Axl when said Axl protein is expressed at the surface    of said mammalian cell.

In a preferred embodiment, said mammalian viable cell is a human cell,preferably a cell naturally expressing the Axl protein receptor.

In a particular embodiment, the mammalian viable cells in step c) aremammalian cells which express recombinant Axl protein(s) at theirsurface.

In an also preferred embodiment, said molecule of interest is acytotoxic molecule (also designated herein as cytoxic or cytostaticagent).

In an also preferred embodiment, said molecule of interest is covalentlylinked to said compound capable of binding the Axl protein using alinker, more preferably a peptidic linker, more preferably a cleavablepeptidic linker, more preferably a linker which can be cleaved bynatural intracellular compounds contained in the mammalian cell,particularly in the cytosol of said mammalian cell.

In an also preferred embodiment, said compound capable of binding theAxl protein is an antibody, or functional binding fragment thereof,which is specifically directed against the Axl protein, or against anepitope thereof located into the Axl EDC domain.

The selection step of e) can be realized by any method known by theperson skilled in the art for the evaluation of the intracellulardelivering or internalization. Assay or test capable of demonstrating orevaluating the presence, absence, or the activity of said compoundcapable of specifically binding the Axl protein, or of said complexformed by said compound and said molecule of interest, or of saidmolecule of interest which is covalently linked to said compound, arewell known by the skilled person (see some examples of such test orassay disclosed hereinafter, without limiting these tests to thesefollowing test examples).

More particularly, these tests or assays can be realized by FACS,Immunofluorescence, flow cytometry, western-blot,cytotoxicity/cytostatic evaluations, etc . . . .

In this aspect, the present invention is also directed to an in vitromethod for the preparation of a cytotoxic or cytostatic complex capableof delivering a cytotoxic compound into a mammalian cell, preferably aviable cell, said method comprising the step of:

-   covalently linked a cytotoxic agent to a compound which is:

i) capable of specifically binding the Axl protein, preferably the humanAxl protein, and

ii) is internalized into a mammalian cell following its binding to saidprotein Axl when said Axl protein is expressed at the surface of saidmammalian cell.

In a preferred embodiment, in said in vitro method for the preparationof a cytotoxic or cytostatic complex, said antigen binding capable ofspecifically binding the Axl protein and internalized into a mammaliancell following its binding to said protein Axl when said Axl protein isexpressed at the surface of said mammalian cell, is an antigen bindingprotein selected by an in vitro method for the screening of a compound,or a binding fragment thereof, capable of delivering or internalizing amolecule of interest into mammalian cells according to the presentinvention.

Preferably said compound is a protein-like protein, more preferably anantibody which is specifically directed against the Axl protein, oragainst an epitope thereof located into the Axl EDC domain, or afunctional binding fragment of said antibody.

In preferred embodiment, said cytotoxic agent is covalently linked tothe said anti-Axl antibody or functional fragment thereof, using alinker, more preferably a peptidic linker, more preferably a cleavablepeptidic linker, more preferably a linker which can be cleaved, as nonlimitative example by natural intracellular compounds.

Like the other members of the TAM family, the Axl extracellular domain(ECD) has an organization closed to those of cell adhesion molecules.Axl ECD is characterized by a combination of two immunoglobulin-likedomains followed by two adjacent fibronectin type III-like domains[O'Bryan J. P. et al. Mol. Cell Biol. (1991).11, 5016-5031]. The twoN-terminal immunoglobulin-like domains are sufficient for Gas6 ligandbinding [Sasaki T. et al. EMBO J. (2006).25, 80-87].

The ECD of the human protein Axl is a 451 amino acids fragment,corresponding to amino acids 1-451 of the sequence SEQ ID NO. 29, whichsequence is represented in the sequence listing as SEQ ID NO. 31. Aminoacids 1-25 corresponding to the signal peptide, the ECD of the humanprotein Axl without the signal peptide corresponds to the amino acids26-451 of the sequence SEQ ID NO.29, represented by the sequence SEQ IDNO. 32.

To date different modes of internalization have been identified. Theyorientate the becoming the internalized proteins or proteic complex inthe cell. After endocytosis, most membranes proteins or lipids returnsto the cell surface (recycling), but some membrane components aredelivered to late endosomes or the Golgi [Maxfield F. R. & McGraw, T. E.Nat. Rev. Mol. Cell Biol. (2004).5, 121-132].

In a preferred embodiment, the invention relates to an antigen bindingprotein, or an antigen binding fragment thereof, which i) specificallybinds to the human protein Axl, and ii) is internalized following itsbinding to said human protein Axl, said antigen binding proteincomprising at least an amino acid sequence selected from the groupconsisting of SEQ ID NOs. 1 to 14, or any sequence exhibiting at least80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID NOs. 1 to 14.

In a most preferred embodiment, the invention relates to an antigenbinding protein, or an antigen binding fragment thereof, which

i) specifically binds to the human protein Axl, preferably having thesequence SEQ ID NO. 29 or 30 or natural variant sequence thereof, and

ii) is internalized following its binding to said human protein Axl,said antigen binding protein comprising at least an amino acid sequenceselected from the group consisting of SEQ ID NOs. 1 to 14.

A “binding protein” or “antigen binding protein” is a peptidic chainhaving a specific or general affinity with another protein or molecule(generally referred as antigen). Proteins are brought into contact andform a complex when binding is possible. The antigen binding protein ofthe invention can preferably be, without limitation, an antibody, afragment or derivative of an antibody, a protein or a peptide.

By “antigen binding fragment” of an antigen binding protein according tothe invention, it is intended to indicate any peptide, polypeptide, orprotein retaining the ability to specifically bind to the target (alsogenerally referred as antigen) of the antigen binding protein andcomprising an amino acid sequence of at least 5 contiguous amino acidresidues, at least 10 contiguous amino acid residues, at least 15contiguous amino acid residues, at least 20 contiguous amino acidresidues, at least 25 contiguous amino acid residues, at least 40contiguous amino acid residues, at least 50 contiguous amino acidresidues, at least 60 contiguous amino residues, at least 70 contiguousamino acid residues, at least contiguous 80 amino acid residues, atleast contiguous 90 amino acid residues, at least contiguous 100 aminoacid residues, at least contiguous 125 amino acid residues, at least 150contiguous amino acid residues, at least contiguous 175 amino acidresidues, at least contiguous 200 amino acid residues, or at leastcontiguous 250 amino acid residues of the amino acid sequence of theantigen binding protein.

In a preferred embodiment wherein the antigen binding protein is anantibody, such “antigen binding fragments” are selected in the groupconsisting of Fv, scFv (sc for single chain), Fab, F(ab′)₂, Fab′,scFv-Fc fragments or diabodies, or any fragment of which the half-lifetime would have been increased by chemical modification, such as theaddition of poly(alkylene) glycol such as poly(ethylene) glycol(“PEGylation”) (pegylated fragments called Fv-PEG, scFv-PEG, Fab-PEG,F(ab′)₂-PEG or Fab′-PEG) (“PEG” for Poly(Ethylene) Glycol), or byincorporation in a liposome, said fragments having at least one of thecharacteristic CDRs of the antibody according to the invention.Preferably, said “antigen binding fragments” will be constituted or willcomprise a partial sequence of the heavy or light variable chain of theantibody from which they are derived, said partial sequence beingsufficient to retain the same specificity of binding as the antibodyfrom which it is descended and a sufficient affinity, preferably atleast equal to 1/100, in a more preferred manner to at least 1/10, ofthe affinity of the antibody from which it is descended, with respect tothe target. Such a functional fragment will contain at the minimum 5amino acids, preferably 10, 15, 25, 50 and 100 consecutive amino acidsof the sequence of the antibody from which it is descended.

The term “epitope” is a region of an antigen that is bound by an antigenbinding protein, including antibodies. Epitopes may be defined asstructural or functional. Functional epitopes are generally a subset ofthe structural epitopes and have those residues that directly contributeto the affinity of the interaction. Epitopes may also be conformational,that is, composed of non-linear amino acids. In certain embodiments,epitopes may include determinants that are chemically active surfacegroupings of molecules such as amino acids, sugar side chains,phosphoryl groups, or sulfonyl groups, and, in certain embodiments, mayhave specific three-dimensional structural characteristics, and/orspecific charge characteristics.

In the present application, the epitope is localized into theextracellular domain of the human protein Axl.

According to a preferred embodiment of the invention, the antigenbinding protein, or an antigen binding fragment thereof, specificallybinds to an epitope localized into the human protein Axl extracellulardomain, preferably having the sequence SEQ ID NO. 31 or 32 or naturalvariant sequence thereof.

By “specifically binding”, “specifically binds”, or the like, it isintended that the antigen binding protein, or antigen-binding fragmentthereof, forms a complex with an antigen that is relatively stable underphysiologic conditions. Specific binding can be characterized by anequilibrium dissociation constant of at least about 1.10⁻⁶ M or less.Methods for determining whether two molecules specifically bind are wellknown in the art and include, for example, equilibrium dialysis, surfaceplasmon resonance, and the like. For the avoidance of doubt, it does notmean that the said antigen binding fragment could not bind or interfere,at a low level, to another antigen. Nevertheless, as a preferredembodiment, the said antigen binding fragment binds only to the saidantigen.

In this sense, “EC₅₀” refers to 50% effective concentration. Moreprecisely the term half maximal effective concentration (EC₅₀)corresponds to the concentration of a drug, antibody or toxicant whichinduces a response halfway between the baseline and maximum after somespecified exposure time. It is commonly used as a measure of drug'spotency. The EC₅₀ of a graded dose response curve therefore representsthe concentration of a compound where 50% of its maximal effect isobserved. The EC₅₀ of a quantal dose response curve represents theconcentration of a compound where 50% of the population exhibits aresponse, after specified exposure duration. Concentration measurestypically follow a sigmoidal curve, increasing rapidly over a relativelysmall change in concentration. This can be determined mathematically byderivation of the best-fit line.

As a preferred embodiment, the EC₅₀ determined in the present inventioncharacterized the potency of antibody binding on the Axl ECD exposed onhuman tumor cells. The EC₅₀ parameter is determined using FACS analysis.The EC₅₀ parameter reflects the antibody concentration for which 50% ofthe maximal binding on the human Axl expressed on human tumor cells isobtained. Each EC₅₀ value was calculated as the midpoint of the doseresponse curve using a four-parameter regression curve fitting program(Prism Software). This parameter has been selected as to berepresentative of physiological/pathological conditions.

In an embodiment of the invention, the antigen binding protein, or anantigen binding fragment thereof, binds to its epitope with an EC₅₀ ofat least 10⁻⁹ M, preferentially between 10⁻⁹ and 10⁻¹² M.

Another embodiment of the invention is a process or method for theselection of an antigen binding protein, or an antigen binding fragmentthereof, capable of being intracellularly internalizing into a mammaliancell, preferably into a human cell, preferably a viable cell, comprisingthe steps of:

-   i) selecting antigen binding protein which specifically binds to    Axl, preferably to its EDC domain or to an epitope thereof; and-   ii) selecting said antigen binding protein from previous step i)    which is internalized into a mammalian cell following their binding    to an Axl protein expressed at the surface of said mammalian cell.

In a particular embodiment, said mammalian cell naturally expresses theAxl protein receptor at their surface or are mammalian cells whichexpress recombinant Axl protein at their surface, preferably humancells.

Such method or process can comprise the steps of i) selecting antigenbinding protein which specifically bind to Axl with an EC⁵⁰ of at least10⁻⁹ M and ii) selecting antigen binding protein from previous stepwhich are internalized following their binding to Axl. The selectionstep of ii) can be realized by any method known by the person skilled inthe art for the evaluation of the internalization. More particularly,tests can be realized by FACS, Immunofluorescence, flow cytometry,western-blot, cytotoxicity evaluations, etc . . . .

Another characteristic of the antigen binding protein according to theinvention is that it does not have any significant activity on theproliferation of tumor cells. More particularly, as illustrated in thefollowing examples, the antigen binding protein according to theinvention does not have any significant in vitro activity on theproliferation SN12C model.

In oncology, there are multiple mechanisms by which mAbs can exerttherapeutic efficacy, but often their activity is not sufficient toproduce a lasting benefit. Hence several strategies have been employedto enhance their activity particularly by combining them with drugs aschemotherapeutic agents. As an efficient alternative to combinationprotocols, immunotoxins become a novel therapeutic option for treatingcancer [Beck A. et al. Discov. Med. (2010).10, 329-339; Alley S. C. etal. J. Pharmacol. Exp. Ther. (2009). 330, 932-938]. Antibody-drugconjugates (ADCs) represent one approach where the ability to harnessmAbs specificity and target the delivery of a cytotoxic agent to thetumor may significantly enhance both mAbs and drug activities. Ideallythe mAb will specifically bind to an antigen with substantial expressionon tumor cells but limited expression on normal cells.

In the present description or figures the terms “Axl antibody” and“anti-Axl antibody” have the same meaning and designate antibodiesdirected against Axl and/or capable of binding specifically the Axlprotein.

The present invention focused on a specific anti-Axl binding protein,and more particularly on a specific anti-Axl antibody, presenting a highability to be internalized following Axl binding. Such antigen bindingprotein is interesting as one of the immuno-drug-conjugate components,so it addresses the linked cytotoxic into the targeted cancer cells.Once internalized the cytotoxic triggers cancer cell death.

Important keys to success with immunoconjugate therapy are thought to bethe target antigen specificity and the internalization of theantigen-binding protein complexes into the cancer cells. Obviouslynon-internalizing antigens are less effective than internalizingantigens to delivers cytotoxic agents. Internalization processes arevariable across antigens and depend on multiple parameters that can beinfluenced by binding proteins. Cell-surface RTKs constitute aninteresting antigens family to investigate for such an approach.

In the biomolecule, the cytotoxic brings the cytotoxic activity and theused antigen binding protein brings its specificity against cancercells, as well as a vector for entering within the cells to correctlyaddress the cytotoxic.

Thus to improve the immunoconjugate molecule, the carrier-bindingprotein must exhibit high ability to internalize into the targetedcancer cells. The efficiency with which the binding proteins mediatedinternalisation differs significantly depending on the epitope targeted.Selection of potent internalizing Axl binding proteins requires variousexperimental data studying not only Axl downregulation but alsofollowing Axl binding proteins becoming into the cells.

In a preferred embodiment, the internalization of the antigen bindingprotein according to the invention can be evaluated preferably byimmunofluorescence (as exemplified hereinafter in the presentapplication) or any method or process known by the person skilled in theart specific for the internalization mechanism.

In another preferred embodiment, as the complex Axl-antigen bindingprotein, according to the invention, is internalized after the bindingof the binding protein of the invention to the ECD of said Axl, areduction in the quantity of Axl at the surface of the cells is induced.This reduction can be quantified by any method known by the personskilled in the art (western-blot, FACS, immunofluorescence, etc . . . ).

In an embodiment of the invention, this reduction, thus reflecting theinternalization, can be preferably measured by FACS and expressed as thedifference or delta between the Mean Fluorescence Intensity (MFI)measured on untreated cells with the MFI measured with cells treatedwith the antigen binding protein according to the invention.

As non limitative example of the present invention, this delta isdetermined based on MFIs obtained with untreated cells and cells treatedwith the antigen binding protein of the invention as described inexample 9 using i) human renal tumor SN12C cells after a 24 hourincubation period with the antigen binding protein of the invention andii) a secondary antibody labelled with Alexa488. This parameter isdefined as calculated with the following formula:Δ(MFI_(24 h) untreated cells−MFI_(24 h) antigen binding protein treatedcells)This difference between MFIs reflects the Axl downregulation as MFIs areproportional of Axl expressed on the cell-surface.

In a more preferred and advantageous aspect, the antigen bindingprotein, or an antigen binding fragment thereof, of the inventionconsists of a monoclonal antibody, preferably an isolated Mab,triggering a Δ(MFI_(24 h) untreated cells−MFI_(24 h) treated cells) ofat least 200, preferably of at least 300.

The antigen binding protein, or an antigen binding fragment thereof,according to the invention, induces a reduction of MFI of at least 200.

In more details, the above mentioned delta can be measured according tothe following process, which must be considered as an illustrative andnon limitative example:

-   -   a) Treating and incubating tumoral cells of interest with the        antigen binding protein of the invention;    -   b) Treating the treated cells of step a) and, in parallel,        untreated cells with the antigen binding protein of the        invention,    -   c) Measuring the MFI (representative of the quantity of Axl        present at the surface) for the treated and the non treated        cells with a secondary labeled antibody capable of binding to        the antigen binding protein, and    -   d) Calculating the delta as the subtraction of the MFI obtained        with the treated cells from the MFI obtained with the non        treated cells.

The terms “antibody”, “antibodies” or “immunoglobulin” are usedinterchangeably in the broadest sense and include monoclonal antibodies,preferably isolated Mab, (e.g., full length or intact monoclonalantibodies), polyclonal antibodies, multivalent antibodies ormultispecific antibodies (e.g., bispecific antibodies so long as theyexhibit the desired biological activity).

More particularly, such molecule consists of a glycoprotein comprisingat least two heavy (H) chains and two light (L) chains inter-connectedby disulfide bonds. Each heavy chain comprises a heavy chain variableregion (or domain) (abbreviated herein as HCVR or VH) and a heavy chainconstant region. The heavy chain constant region comprises threedomains, CH1, CH2 and CH3. Each light chain comprises a light chainvariable region (abbreviated herein as LCVR or VL) and a light chainconstant region. The light chain constant region comprises one domain,CL. The VH and VL regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR). Each VH and VL is composed of three CDRs and four FRs,arranged from amino-terminus to carboxy-terminus in the following order:FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavyand light chains contain a binding domain that interacts with anantigen. The constant regions of the antibodies may mediate the bindingof the immunoglobulin to host tissues or factors, including variouscells of the immune system (e.g. effector cells) and the first component(Clq) of the classical complement system.

Antibodies in the sense of the invention also include certain antibodyfragments, thereof. The said antibody fragments exhibit the desiredbinding specificity and affinity, regardless of the source orimmunoglobulin type (i.e., IgG, IgE, IgM, IgA, etc.), i.e., they arecapable of binding specifically the Axl protein with an affinitycomparable to the full-length antibodies of the invention.

In general, for the preparation of monoclonal antibodies or theirfunctional fragments, especially of murine origin, it is possible torefer to techniques which are described in particular in the manual“Antibodies” (Harlow and Lane, Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory, Cold Spring Harbor N.Y., pp. 726, 1988) or tothe technique of preparation from hybridomas described by Kohler andMilstein (Nature, 256:495-497, 1975).

The term “monoclonal antibody” or “Mab” as used herein refers to anantibody molecule that is directed against a specific antigen and whichmay be produced by a single clone of B cells or hybridoma. Monoclonalantibodies may also be recombinant, i.e. produced by proteinengineering. In addition, in contrast with preparations of polyclonalantibodies which typically include various antibodies directed againstvarious determinants, or epitopes, each monoclonal antibody is directedagainst a single epitope of the antigen. The invention relates toantibodies isolated or obtained by purification from natural sources orobtained by genetic recombination or chemical synthesis.

An embodiment of the invention is an antigen binding protein, or anantigen binding fragment thereof, comprising or consisting of anantibody selected in the group consisting of 1A4, 11D12, 320G10, 427B7,517F1, 530C8, 534G9, 535F7, 547A1, 1023F10, 1613F12 or 1614G5.

A preferred embodiment of the invention is an antigen binding protein,or an antigen binding fragment thereof, comprising or consisting of anantibody, said antibody comprising the three light chain CDRs comprisingthe sequences SEQ ID NOs. 1, 2 and 3, or any sequence exhibiting atleast 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID NOs. 1,2 and 3; and the three heavy chain CDRs comprising the sequences SEQ IDNOs. 4, 5 and 6, or any sequence exhibiting at least 80%, preferably85%, 90%, 95% and 98% identity with SEQ ID NOs. 4, 5 and 6.

In a more preferred embodiment of the invention, the antigen bindingprotein, or an antigen binding fragment thereof, consists of anantibody, said antibody comprising the three light chain CDRs comprisingthe sequences SEQ ID NOs. 1, 2 and 3; and the three heavy chain CDRscomprising the sequences SEQ ID NOs. 4, 5 and 6.

In a preferred aspect, by CDR regions or CDR(s), it is intended toindicate the hypervariable regions of the heavy and light chains of theimmunoglobulins as defined by IMGT. Without any contradictory mention,the CDRs will be defined in the present specification according to theIMGT numbering system.

The IMGT unique numbering has been defined to compare the variabledomains whatever the antigen receptor, the chain type, or the species[Lefranc M.-P., Immunology Today 18, 509 (1997)/Lefranc M.-P., TheImmunologist, 7, 132-136 (1999)/Lefranc, M.-P., Pommié, C., Ruiz, M.,Giudicelli, V., Foulquier, E., Truong, L., Thouvenin-Contet, V. andLefranc, Dev. Comp. Immunol., 27, 55-77 (2003)]. In the IMGT uniquenumbering, the conserved amino acids always have the same position, forinstance cystein 23 (1st-CYS), tryptophan 41 (CONSERVED-TRP),hydrophobic amino acid 89, cystein 104 (2nd-CYS), phenylalanine ortryptophan 118 (J-PHE or J-TRP). The IMGT unique numbering provides astandardized delimitation of the framework regions (FR1-IMGT: positions1 to 26, FR2-IMGT: 39 to 55, FR3-IMGT: 66 to 104 and FR4-IMGT: 118 to128) and of the complementarity determining regions: CDR1-IMGT: 27 to38, CDR2-IMGT: 56 to 65 and CDR3-IMGT: 105 to 117. As gaps representunoccupied positions, the CDR-IMGT lengths (shown between brackets andseparated by dots, e.g. [8.8.13]) become crucial information. The IMGTunique numbering is used in 2D graphical representations, designated asIMGT Colliers de Perles [Ruiz, M. and Lefranc, M.-P., Immunogenetics,53, 857-883 (2002)/Kaas, Q. and Lefranc, M.-P., Current Bioinformatics,2, 21-30 (2007)], and in 3D structures in IMGT/3Dstructure-DB [Kaas, Q.,Ruiz, M. and Lefranc, M.-P., T cell receptor and MHC structural data.Nucl. Acids. Res., 32, D208-D210 (2004)].

It must be understood that, without contradictory specification in thepresent specification, complementarity-determining regions or CDRs, meanthe hypervariable regions of the heavy and light chains ofimmunoglobulins as defined according to the IMGT numbering system.

Nevertheless, CDRs can also be defined according to the Kabat numberingsystem (Kabat et al., Sequences of proteins of immunological interest,5^(th) Ed., U.S. Department of Health and Human Services, NIH, 1991, andlater editions). There are three heavy-chain CDRs and three light-chainCDRs. Here, the terms “CDR” and “CDRs” are used to indicate, dependingon the case, one or more, or even all, of the regions containing themajority of the amino acid residues responsible for the antibody'sbinding affinity for the antigen or epitope it recognizes.

According to the Kabat numbering system, the present invention relatesto an antigen binding protein, or an antigen binding fragment thereof,consisting of an antibody, said antibody comprising the three lightchain CDRs, as defined according to Kabat numbering system, comprisingthe sequences SEQ ID NOs. 9, 10 and 11, or any sequence exhibiting atleast 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID NOs. 9,10 and 11; and the three heavy chain CDRs, as defined according to Kabatnumbering system, comprising the sequences SEQ ID NOs. 12, 13 and 14, orany sequence exhibiting at least 80%, preferably 85%, 90%, 95% and 98%identity with SEQ ID NOs. 12, 13 and 14.

In the sense of the present invention, the “percentage identity” betweentwo sequences of nucleic acids or amino acids means the percentage ofidentical nucleotides or amino acid residues between the two sequencesto be compared, obtained after optimal alignment, this percentage beingpurely statistical and the differences between the two sequences beingdistributed randomly along their length. The comparison of two nucleicacid or amino acid sequences is traditionally carried out by comparingthe sequences after having optimally aligned them, said comparison beingable to be conducted by segment or by using an “alignment window”.Optimal alignment of the sequences for comparison can be carried out, inaddition to comparison by hand, by means of the local homology algorithmof Smith and Waterman (1981) [Ad. App. Math. 2:482], by means of thelocal homology algorithm of Neddleman and Wunsch (1970) [J. Mol. Biol.48:443], by means of the similarity search method of Pearson and Lipman(1988) [Proc. Natl. Acad. Sci. USA 85:2444] or by means of computersoftware using these algorithms (GAP, BESTFIT, FASTA and TFASTA in theWisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis., or by the comparison software BLAST NR orBLAST P).

The percentage identity between two nucleic acid or amino acid sequencesis determined by comparing the two optimally-aligned sequences in whichthe nucleic acid or amino acid sequence to compare can have additions ordeletions compared to the reference sequence for optimal alignmentbetween the two sequences. Percentage identity is calculated bydetermining the number of positions at which the amino acid nucleotideor residue is identical between the two sequences, preferably betweenthe two complete sequences, dividing the number of identical positionsby the total number of positions in the alignment window and multiplyingthe result by 100 to obtain the percentage identity between the twosequences.

For example, the BLAST program, “BLAST 2 sequences” (Tatusova et al.,“Blast 2 sequences—a new tool for comparing protein and nucleotidesequences”, FEMS Microbiol., 1999, Lett. 174:247-250) available on thesite http://www.ncbi.nlm.nih.gov/gorf/b12.html, can be used with thedefault parameters (notably for the parameters “open gap penalty”: 5,and “extension gap penalty”: 2; the selected matrix being for examplethe “BLOSUM 62” matrix proposed by the program); the percentage identitybetween the two sequences to compare is calculated directly by theprogram.

For the amino acid sequence exhibiting at least 80%, preferably 85%,90%, 95% and 98% identity with a reference amino acid sequence,preferred examples include those containing the reference sequence,certain modifications, notably a deletion, addition or substitution ofat least one amino acid, truncation or extension. In the case ofsubstitution of one or more consecutive or non-consecutive amino acids,substitutions are preferred in which the substituted amino acids arereplaced by “equivalent” amino acids. Here, the expression “equivalentamino acids” is meant to indicate any amino acids likely to besubstituted for one of the structural amino acids without howevermodifying the biological activities of the corresponding antibodies andof those specific examples defined below.

Equivalent amino acids can be determined either on their structuralhomology with the amino acids for which they are substituted or on theresults of comparative tests of biological activity between the variousantigen binding proteins likely to be generated.

As a non-limiting example, table 2 below summarizes the possiblesubstitutions likely to be carried out without resulting in asignificant modification of the biological activity of the correspondingmodified antigen binding protein; inverse substitutions are naturallypossible under the same conditions.

TABLE 2 Original residue Substitution(s) Ala (A) Val, Gly, Pro Arg (R)Lys, His Asn (N) Gln Asp (D) Glu Cys (C) Ser Gln (Q) Asn Glu (E) Asp Gly(G) Ala His (H) Arg Ile (I) Leu Leu (L) Ile, Val, Met Lys (K) Arg Met(M) Leu Phe (F) Tyr Pro (P) Ala Ser (S) Thr, Cys Thr (T) Ser Trp (W) TyrTyr (Y) Phe, Trp Val (V) Leu, Ala

An embodiment of the invention relates to an antigen binding protein, oran antigen binding fragment thereof, comprising the three light chainCDRs comprising the sequences SEQ ID NOs. 1, 2 and 3, or any sequenceexhibiting at least 80%, preferably 85%, 90%, 95% and 98% identity withSEQ ID NOs.1, 2 and 3; and a heavy chain variable domain of sequence SEQID NO. 8, or any sequence exhibiting at least 80%, preferably 85%, 90%,95% and 98% identity with SEQ ID NO. 8.

According to a preferred embodiment of the invention, the antigenbinding protein, or an antigen binding fragment thereof comprises thethree light chain CDRs comprising the sequences SEQ ID NOs. 1, 2 and 3;and a heavy chain variable domain of sequence SEQ ID NO. 8, or anysequence exhibiting at least 80% identity with SEQ ID NO.8.

According to another preferred embodiment of the invention, the antigenbinding protein, or an antigen binding fragment thereof comprises aheavy chain variable domain of sequence SEQ ID NO. 8, or any sequenceexhibiting at least 80% identity with SEQ ID NO.8.

By “any sequence exhibiting at least 80% identity with SEQ ID NO. 8”,its is intended to designate the sequence exhibiting the three heavychain CDRs SEQ ID NOs. 4, 5 and 6 and, in addition, exhibiting at least80% identity with the full sequence SEQ ID NO. 8 outside the sequencescorresponding to the CDRs, i.e. SEQ ID NOs. 4, 5 and 6.

Another embodiment of the invention relates to an antigen bindingprotein, or an antigen binding fragment thereof, comprising a lightchain variable domain of sequence SEQ ID NO. 7, or any sequenceexhibiting at least 80%, preferably 85%, 90%, 95% and 98% identity withSEQ ID NO. 7; and the three heavy chain CDRs comprising the sequencesSEQ ID NOs. 4, 5 and 6, or any sequence exhibiting at least 80%,preferably 85%, 90%, 95% and 98% identity with SEQ ID NOs. 4, 5 and 6.

According to a preferred embodiment of the invention, the antigenbinding protein, or an antigen binding fragment thereof, comprises alight chain variable domain of sequence SEQ ID NO. 7, or any sequenceexhibiting at least 80% identity with SEQ ID NO.7; and the three heavychain CDRs comprising the sequences SEQ ID NOs. 4, 5 and 6.

According to another preferred embodiment of the invention, the antigenbinding protein, or an antigen binding fragment thereof, comprises alight chain variable domain of sequence SEQ ID NO. 7, or any sequenceexhibiting at least 80% identity with SEQ ID NO.7.

By “any sequence exhibiting at least 80% identity with SEQ ID NO. 7”,its is also intended to designate the sequence exhibiting the threelight chain CDRs SEQ ID NOs. 1, 2 and 3 and, in addition, exhibiting atleast 80% identity with the full sequence SEQ ID NO. 7 outside thesequences corresponding to the CDRs, i.e. SEQ ID NOs. 1, 2 and 3.

Another embodiment of the invention relates to an antigen bindingprotein, or an antigen binding fragment thereof, comprising a lightchain variable domain of sequence SEQ ID NO. 7, or any sequenceexhibiting at least 80%, preferably 85%, 90%, 95% and 98% identity withSEQ ID NO. 7; and a heavy chain variable domain of sequence SEQ ID NO.8, or any sequence exhibiting at least 80%, preferably 85%, 90%, 95% and98% identity with SEQ ID NO. 8.

According to a preferred embodiment of the invention, the antigenbinding protein, or an antigen binding fragment thereof, comprises alight chain variable domain of sequence SEQ ID NO. 7, or any sequenceexhibiting at least 80% identity with SEQ ID NO. 7 and a heavy chainvariable domain of sequence SEQ ID NO. 8, or any sequence exhibiting atleast 80% identity with SEQ ID NO. 8.

For more clarity, table 3 below summarizes the various amino acidsequences corresponding to the antigen binding protein of the invention(with Mu.=murine).

TABLE 3 CDR SEQ numbering Heavy chain Light chain ID NO. 1613F12 IMGTCDR-L1 1 CDR-L2 2 CDR-L3 3 CDR-H1 4 CDR-H2 5 CDR-H3 6 Kabat CDR-L1 9CDR-L2 10 CDR-L3 11 CDR-H1 12 CDR-H2 13 CDR-H3 14 Mu. variable 7 domainMu. variable domain 8

A specific aspect of the present invention relates to a murine antibody,or its derived compounds or antigen binding fragments, characterized inthat said antibody also comprises light-chain and heavy-chain constantregions derived from an antibody of a species heterologous with themouse, notably man.

Another specific aspect of the present invention relates to a chimericantibody, or its derived compounds or antigen binding fragments,characterized in that said antibody also comprises light-chain andheavy-chain constant regions derived from an antibody of a speciesheterologous with the mouse, notably human.

Yet another specific aspect of the present invention relates to ahumanized antibody, or its derived compounds or antigen bindingfragments, characterized in that the constant regions of the light-chainand the heavy-chain derived from human antibody are, respectively, thelambda or kappa region and the gamma-1, gamma-2 or gamma-4 region.

Another aspect of the invention is an antigen binding protein consistingof the monoclonal antibody 1613F12 derived from the hybridoma I-4505deposited at the CNCM, Institute Pasteur, France, on the 28 Jul. 2011,or an antigen binding fragment thereof.

According to another aspect, the invention relates to a murine hybridomacapable of secreting an antigen binding protein according to theinvention, notably the hybridoma of murine origin filed with the Frenchcollection for microorganism cultures (CNCM, Pasteur Institute, Paris,France) on Jul. 28, 2011, under number I-4505. Said hybridoma wasobtained by the fusion of Balb/C immunized mice splenocytes/lymphocytesand cells of the myeloma Sp 2/O—Ag 14 cell line.

According to another aspect, the invention relates to a murine hybridomacapable of secreting an antibody comprising the three light chain CDRscomprising the sequences SEQ ID NOs. 1, 2 and 3; and the three heavychain CDRs comprising the sequences SEQ ID NOs. 4, 5 and 6, saidhybridoma being filed at the CNCM, Pasteur Institute, Paris, France, onJul. 28, 2011, under number I-4505. Said hybridoma was obtained by thefusion of Balb/C immunized mice splenocytes/lymphocytes and cells of themyeloma Sp 2/O—Ag 14 cell line.

An object of the invention is the murine hybridoma I-4505 deposited atthe CNCM, Institute Pasteur, France, on the 28 Jul. 2011.

Another aspect of the invention is an antigen binding protein consistingof the monoclonal antibody 1A4 derived from the hybridoma I-3947deposited at the CNCM, Institute Pasteur, France, on Mar. 12, 2008, oran antigen binding fragment thereof.

According to another aspect, the invention relates to a murine hybridomacapable of secreting an antigen binding protein according to theinvention, notably the hybridoma of murine origin filed with the Frenchcollection for microorganism cultures (CNCM, Pasteur Institute, Paris,France) on Mar. 12, 2008, under number I-3947. Said hybridoma wasobtained by the fusion of Balb/C immunized mice splenocytes/lymphocytesand cells of the myeloma Sp 2/O—Ag 14 cell line.

Another aspect of the invention is an antigen binding protein consistingof the monoclonal antibody 11D12 derived from the hybridoma I-4116deposited at the CNCM, Institute Pasteur, France, on Jan. 27, 2009, oran antigen binding fragment thereof.

According to another aspect, the invention relates to a murine hybridomacapable of secreting an antigen binding protein according to theinvention, notably the hybridoma of murine origin filed with the Frenchcollection for microorganism cultures (CNCM, Pasteur Institute, Paris,France) on Jan. 27, 2009, under number I-4116. Said hybridoma wasobtained by the fusion of Balb/C immunized mice splenocytes/lymphocytesand cells of the myeloma Sp 2/O—Ag 14 cell line.

Another aspect of the invention is an antigen binding protein consistingof the monoclonal antibody 320G10 derived from the hybridoma I-4514deposited at the CNCM, Institute Pasteur, France, on Sep. 15, 2011, oran antigen binding fragment thereof.

According to another aspect, the invention relates to a murine hybridomacapable of secreting an antigen binding protein according to theinvention, notably the hybridoma of murine origin filed with the Frenchcollection for microorganism cultures (CNCM, Pasteur Institute, Paris,France) on Sep. 15, 2011, under number I-4514. Said hybridoma wasobtained by the fusion of Balb/C immunized mice splenocytes/lymphocytesand cells of the myeloma Sp 2/O—Ag 14 cell line.

Another aspect of the invention is an antigen binding protein consistingof the monoclonal antibody 427B7 derived from the hybridoma I-4518deposited at the CNCM, Institute Pasteur, France, on Sep. 15, 2011, oran antigen binding fragment thereof.

According to another aspect, the invention relates to a murine hybridomacapable of secreting an antigen binding protein according to theinvention, notably the hybridoma of murine origin filed with the Frenchcollection for microorganism cultures (CNCM, Pasteur Institute, Paris,France) on Sep. 15, 2011, under number I-4518. Said hybridoma wasobtained by the fusion of Balb/C immunized mice splenocytes/lymphocytesand cells of the myeloma Sp 2/O—Ag 14 cell line.

Another aspect of the invention is an antigen binding protein consistingof the monoclonal antibody 517F1 derived from the hybridoma I-4502deposited at the CNCM, Institute Pasteur, France, on Jul. 28, 2011, oran antigen binding fragment thereof.

According to another aspect, the invention relates to a murine hybridomacapable of secreting an antigen binding protein according to theinvention, notably the hybridoma of murine origin filed with the Frenchcollection for microorganism cultures (CNCM, Pasteur Institute, Paris,France) on Jul. 28, 2011, under number I-4502. Said hybridoma wasobtained by the fusion of Balb/C immunized mice splenocytes/lymphocytesand cells of the myeloma Sp 2/O—Ag 14 cell line.

Another aspect of the invention is an antigen binding protein consistingof the monoclonal antibody 530C8 derived from the hybridoma I-4728deposited at the CNCM, Institute Pasteur, France, on Apr. 4, 2013, or anantigen binding fragment thereof.

According to another aspect, the invention relates to a murine hybridomacapable of secreting an antigen binding protein according to theinvention, notably the hybridoma of murine origin filed with the Frenchcollection for microorganism cultures (CNCM, Pasteur Institute, Paris,France) on Apr. 4, 2013, under number I-4728. Said hybridoma wasobtained by the fusion of Balb/C immunized mice splenocytes/lymphocytesand cells of the myeloma Sp 2/O—Ag 14 cell line.

Another aspect of the invention is an antigen binding protein consistingof the monoclonal antibody 534G9 derived from the hybridoma I-4729deposited at the CNCM, Institute Pasteur, France, on Apr., 4, 2013, oran antigen binding fragment thereof.

According to another aspect, the invention relates to a murine hybridomacapable of secreting an antigen binding protein according to theinvention, notably the hybridoma of murine origin filed with the Frenchcollection for microorganism cultures (CNCM, Pasteur Institute, Paris,France) on Apr. 4, 2013, under number I-4729. Said hybridoma wasobtained by the fusion of Balb/C immunized mice splenocytes/lymphocytesand cells of the myeloma Sp 2/O—Ag 14 cell line.

Another aspect of the invention is an antigen binding protein consistingof the monoclonal antibody 535F7 derived from the hybridoma I-4510deposited at the CNCM, Institute Pasteur, France, on Jul. 28, 2011, oran antigen binding fragment thereof.

According to another aspect, the invention relates to a murine hybridomacapable of secreting an antigen binding protein according to theinvention, notably the hybridoma of murine origin filed with the Frenchcollection for microorganism cultures (CNCM, Pasteur Institute, Paris,France) on Jul. 28, 2011, under number I-4510. Said hybridoma wasobtained by the fusion of Balb/C immunized mice splenocytes/lymphocytesand cells of the myeloma Sp 2/O—Ag 14 cell line.

Another aspect of the invention is an antigen binding protein consistingof the monoclonal antibody 547A1 derived from the hybridoma I-4506deposited at the CNCM, Institute Pasteur, France, on Jul. 28, 2011, oran antigen binding fragment thereof.

According to another aspect, the invention relates to a murine hybridomacapable of secreting an antigen binding protein according to theinvention, notably the hybridoma of murine origin filed with the Frenchcollection for microorganism cultures (CNCM, Pasteur Institute, Paris,France) on Jul. 28, 2011, under number I-4506. Said hybridoma wasobtained by the fusion of Balb/C immunized mice splenocytes/lymphocytesand cells of the myeloma Sp 2/O—Ag 14 cell line.

Another aspect of the invention is an antigen binding protein consistingof the monoclonal antibody 1023F10 derived from the hybridoma I-4507deposited at the CNCM, Institute Pasteur, France, on Jul. 28, 2011, oran antigen binding fragment thereof.

According to another aspect, the invention relates to a murine hybridomacapable of secreting an antigen binding protein according to theinvention, notably the hybridoma of murine origin filed with the Frenchcollection for microorganism cultures (CNCM, Pasteur Institute, Paris,France) on Jul. 28, 2011, under number I-4507. Said hybridoma wasobtained by the fusion of Balb/C immunized mice splenocytes/lymphocytesand cells of the myeloma Sp 2/O—Ag 14 cell line.

Another aspect of the invention is an antigen binding protein consistingof the monoclonal antibody 1614G5 derived from the hybridoma I-4519deposited at the CNCM, Institute Pasteur, France, on Sep. 15, 2011, oran antigen binding fragment thereof.

According to another aspect, the invention relates to a murine hybridomacapable of secreting an antigen binding protein according to theinvention, notably the hybridoma of murine origin filed with the Frenchcollection for microorganism cultures (CNCM, Pasteur Institute, Paris,France) on Sep. 15, 2011, under number I-4519. Said hybridoma wasobtained by the fusion of Balb/C immunized mice splenocytes/lymphocytesand cells of the myeloma Sp 2/O—Ag 14 cell line.

The antigen binding protein of the invention also comprises chimeric orhumanized antibodies.

A chimeric antibody is one containing a natural variable region (lightchain and heavy chain) derived from an antibody of a given species incombination with constant regions of the light chain and the heavy chainof an antibody of a species heterologous to said given species.

The antibodies, or chimeric fragments of same, can be prepared by usingthe techniques of recombinant genetics. For example, the chimericantibody could be produced by cloning recombinant DNA containing apromoter and a sequence coding for the variable region of a nonhumanmonoclonal antibody of the invention, notably murine, and a sequencecoding for the human antibody constant region. A chimeric antibodyaccording to the invention coded by one such recombinant gene could be,for example, a mouse-human chimera, the specificity of this antibodybeing determined by the variable region derived from the murine DNA andits isotype determined by the constant region derived from human DNA.Refer to Verhoeyn et al. (BioEssays, 8:74, 1988) for methods forpreparing chimeric antibodies.

In another aspect, the invention describes a binding protein whichconsists of a chimeric antibody.

In a particular preferred embodiment, the chimeric antibody, or anantigen binding fragment of same, of the invention comprises a lightchain variable domain sequence comprising the amino acid sequence SEQ IDNO. 7, and in that it comprises a heavy chain variable domain sequencecomprising the amino acid sequence SEQ ID NO. 8.

In another aspect, the invention describes a binding protein whichconsists of a humanized antibody.

“Humanized antibodies” means an antibody that contains CDR regionsderived from an antibody of nonhuman origin, the other parts of theantibody molecule being derived from one (or several) human antibodies.In addition, some of the skeleton segment residues (called FR) can bemodified to preserve binding affinity (Jones et al., Nature,321:522-525, 1986; Verhoeyen et al., Science, 239:1534-1536, 1988;Riechmann et al., Nature, 332:323-327, 1988).

The humanized antibodies of the invention or fragments of same can beprepared by techniques known to a person skilled in the art (such as,for example, those described in the documents Singer et al., J. Immun.,150:2844-2857, 1992; Mountain et al., Biotechnol. Genet. Eng. Rev.,10:1-142, 1992; and Bebbington et al., Bio/Technology, 10:169-175,1992). Such humanized antibodies are preferred for their use in methodsinvolving in vitro diagnoses or preventive and/or therapeutic treatmentin vivo. Other humanization techniques, also known to a person skilledin the art, such as, for example, the “CDR grafting” technique describedby PDL in patents EP 0 451 261, EP 0 682 040, EP 0 939 127, EP 0 566 647or U.S. Pat. No. 5,530,101, U.S. Pat. No. 6,180,370, U.S. Pat. No.5,585,089 and U.S. Pat. No. 5,693,761. U.S. Pat. No. 5,639,641 or6,054,297, 5,886,152 and 5,877,293 can also be cited.

In addition, the invention also relates to humanized antibodies arisingfrom the murine antibodies described above.

In a preferred manner, constant regions of the light-chain and theheavy-chain derived from human antibody are, respectively, the lambda orkappa and the gamma-1, gamma-2 or gamma-4 region.

A novel aspect of the present invention relates to an isolated nucleicacid characterized in that it is selected among the following nucleicacids (including any degenerate genetic code):

a) a nucleic acid coding for an antigen binding protein, or for anantigen binding fragment of same, according to the invention;

b) a nucleic acid comprising:

a nucleic acid sequence selected from the group consisting of SEQ IDNOs. 15 to 28, or

a nucleic acid sequence comprising the 6 nucleic acid sequences SEQ IDNOs.: 15 to 20, or

a nucleic acid sequence comprising the two nucleic acid sequences SEQ IDNOs.: 21, 22;

c) a nucleic acid complementary to a nucleic acid as defined in a) orb); and

d) a nucleic acid, preferably having at least 18 nucleotides, capable ofhybridizing under highly stringent conditions with a nucleic acidsequence as defined in part a) or b), or with a sequence with at least80%, preferably 85%, 90%, 95% and 98% identity after optimal alignmentwith a nucleic acid sequence as defined in part a) or b).

Table 4 below summarizes the various nucleotide sequences concerning thebinding protein of the invention (with Mu.=Murine).

TABLE 4 CDR SEQ numbering Heavy chain Light chain ID NO. 1613F12 IMGTCDR-L1 15 CDR-L2 16 CDR-L3 17 CDR-H1 18 CDR-H2 19 CDR-H3 20 Kabat CDR-L123 CDR-L2 24 CDR-L3 25 CDR-H1 26 CDR-H2 27 CDR-H3 28 Mu. variable 21domain Mu. variable domain 22

The terms “nucleic acid”, “nucleic sequence”, “nucleic acid sequence”,“polynucleotide”, “oligonucleotide”, “polynucleotide sequence” and“nucleotide sequence”, used interchangeably in the present description,mean a precise sequence of nucleotides, modified or not, defining afragment or a region of a nucleic acid, containing unnatural nucleotidesor not, and being either a double-strand DNA, a single-strand DNA ortranscription products of said DNAs.

The sequences of the present invention have been isolated and/orpurified, i.e., they were sampled directly or indirectly, for example bya copy, their environment having been at least partially modified.Isolated nucleic acids obtained by recombinant genetics, by means, forexample, of host cells, or obtained by chemical synthesis should also bementioned here.

“Nucleic sequences exhibiting a percentage identity of at least 80%,preferably 85%, 90%, 95% and 98%, after optimal alignment with apreferred sequence” means nucleic sequences exhibiting, with respect tothe reference nucleic sequence, certain modifications such as, inparticular, a deletion, a truncation, an extension, a chimeric fusionand/or a substitution, notably punctual. Preferably, these are sequenceswhich code for the same amino acid sequences as the reference sequence,this being related to the degeneration of the genetic code, orcomplementarity sequences that are likely to hybridize specifically withthe reference sequences, preferably under highly stringent conditions,notably those defined below.

Hybridization under highly stringent conditions means that conditionsrelated to temperature and ionic strength are selected in such a waythat they allow hybridization to be maintained between twocomplementarity DNA fragments. On a purely illustrative basis, thehighly stringent conditions of the hybridization step for the purpose ofdefining the polynucleotide fragments described above are advantageouslyas follows.

DNA-DNA or DNA-RNA hybridization is carried out in two steps: (1)prehybridization at 42° C. for three hours in phosphate buffer (20 mM,pH 7.5) containing 5×SSC (1×SSC corresponds to a solution of 0.15 MNaCl+0.015 M sodium citrate), 50% formamide, 7% sodium dodecyl sulfate(SDS), 10×Denhardt's, 5% dextran sulfate and 1% salmon sperm DNA; (2)primary hybridization for 20 hours at a temperature depending on thelength of the probe (i.e.: 42° C. for a probe >100 nucleotides inlength) followed by two 20-minute washings at 20° C. in 2×SSC+2% SDS,one 20-minute washing at 20° C. in 0.1×SSC+0.1% SDS. The last washing iscarried out in 0.1×SSC+0.1% SDS for 30 minutes at 60° C. for aprobe >100 nucleotides in length. The highly stringent hybridizationconditions described above for a polynucleotide of defined size can beadapted by a person skilled in the art for longer or shorteroligonucleotides, according to the procedures described in Sambrook, etal. (Molecular cloning: a laboratory manual, Cold Spring HarborLaboratory; 3rd edition, 2001).

The invention also relates to a vector comprising a nucleic acid asdescribed in the invention.

The invention notably targets cloning and/or expression vectors thatcontain such a nucleotide sequence.

The vectors of the invention preferably contain elements which allow theexpression and/or the secretion of nucleotide sequences in a given hostcell. The vector thus must contain a promoter, translation initiationand termination signals, as well as suitable transcription regulationregions. It must be able to be maintained in a stable manner in the hostcell and may optionally have specific signals which specify secretion ofthe translated protein. These various elements are selected andoptimized by a person skilled in the art according to the host cellused. For this purpose, the nucleotide sequences can be inserted inself-replicating vectors within the chosen host or be integrativevectors of the chosen host.

Such vectors are prepared by methods typically used by a person skilledin the art and the resulting clones can be introduced into a suitablehost by standard methods such as lipofection, electroporation, heatshock or chemical methods.

The vectors are, for example, vectors of plasmid or viral origin. Theyare used to transform host cells in order to clone or express thenucleotide sequences of the invention.

The invention also comprises isolated host cells transformed by orcomprising a vector as described in the present invention.

The host cell can be selected among prokaryotic or eukaryotic systemssuch as bacterial cells, for example, but also yeast cells or animalcells, notably mammal cells (with the exception of human). Insect orplant cells can also be used.

The invention also relates to animals, other than human, that have atransformed cell according to the invention.

Another aspect of the invention relates to a method for the productionof an antigen binding protein according to the invention, or an antigenbinding fragment thereof, characterized in that said method comprisesthe following steps:

a) the culture in a medium with the suitable culture conditions for ahost cell according to the invention; and

b) the recovery of the antigen binding protein, or one of its antigenbinding fragments, thus produced from the culture medium or from saidcultured cells.

The transformed cells according to the invention are of use in methodsfor the preparation of recombinant antigen binding proteins according tothe invention. Methods for the preparation of antigen binding proteinsaccording to the invention in recombinant form, characterized in thatsaid methods use a vector and/or a cell transformed by a vectoraccording to the invention, are also comprised in the present invention.Preferably, a cell transformed by a vector according to the invention iscultured under conditions that allow the expression of the aforesaidantigen binding protein and recovery of said recombinant protein.

As already mentioned, the host cell can be selected among prokaryotic oreukaryotic systems. In particular, it is possible to identify thenucleotide sequences of the invention that facilitate secretion in sucha prokaryotic or eukaryotic system. A vector according to the inventioncarrying such a sequence can thus be used advantageously for theproduction of recombinant proteins to be secreted. Indeed, thepurification of these recombinant proteins of interest will befacilitated by the fact that they are present in the supernatant of thecellular culture rather than inside host cells.

The antigen binding protein of the invention can also be prepared bychemical synthesis. One such method of preparation is also an object ofthe invention. A person skilled in the art knows methods for chemicalsynthesis, such as solid-phase techniques (see notably Steward et al.,1984, Solid phase peptides synthesis, Pierce Chem. Company, Rockford,111, 2nd ed., pp 71-95) or partial solid-phase techniques, bycondensation of fragments or by conventional synthesis in solution.Polypeptides obtained by chemical synthesis and capable of containingcorresponding unnatural amino acids are also comprised in the invention.

The antigen binding protein, or the antigen binding fragments of same,likely to be obtained by the method of the invention are also comprisedin the present invention.

According to a particular aspect, the invention concerns an antigenbinding protein, or an antigen binding fragment thereof, as abovedescribed for use as an addressing product for delivering a cytotoxicagent at a host target site, said host target site consisting of anepitope localized into the protein Axl extracellular domain, preferablythe human protein Axl extracellular domain, more preferably the humanprotein Axl extracellular domain having the sequence SEQ ID NO. 31 or32, or natural variant sequence thereof.

In a preferred embodiment, said host target site is a target site of amammalian cell, more preferably of a human cell, more preferably cellswhich naturally or by way of genetical recombination, express the Axlprotein.

The invention relates to an immunoconjugate comprising the antigenbinding protein as described in the present specification conjugated toa cytotoxic agent.

In the sense of the present invention, the expression “immunoconjugate”or “immuno-conjugate” refers generally to a compound comprising at leastan addressing product physically linked with a one or more therapeuticagent(s), thus creating a highly targeted compound.

In a preferred embodiment, such therapeutic agents consist of cytotoxicagents.

By “cytotoxic agent” or “cytotoxic”, it is intended an agent which, whenadministered to a subject, treats or prevents the development of cellproliferation, preferably the development of cancer in the subject'sbody, by inhibiting or preventing a cellular function and/or causingcell death.

Many cytotoxic agents have been isolated or synthesized and make itpossible to inhibit the cells proliferation, or to destroy or reduce, ifnot definitively, at least significantly the tumour cells. However, thetoxic activity of these agents is not limited to tumour cells, and thenon-tumour cells are also effected and can be destroyed. Moreparticularly, side effects are observed on rapidly renewing cells, suchas haematopoietic cells or cells of the epithelium, in particular of themucous membranes. By way of illustration, the cells of thegastrointestinal tract are largely effected by the use of such cytotoxicagents.

One of the aims of the present invention is also to be able to provide acytotoxic agent which makes it possible to limit the side effects onnormal cells while at the same time conserving a high cytotoxicity ontumour cells.

More particularly, the cytotoxic agent may preferably consist of,without limitation, a drug (i.e “antibody-drug conjugate”), a toxin(i.e. “immunotoxin” or “antibody-toxin conjugate”), a radioisotope (i.e.“radioimmunoconjugate” or “antibody-radioisotope conjugate”), etc.

In a first preferred embodiment of the invention, the immunoconjugateconsists of a binding protein linked to at least a drug or a medicament.Such an immunoconjugate is referred as an antibody-drug conjugate (or“ADC”) when the binding protein is an antibody, or an antigen bindingfragment thereof.

In a first embodiment, such drugs can be described regarding their modeof action. As non limitative example, it can be mentioned alkylatingagents such as nitrogen mustard, alkyle-sulfonates, nitrosourea,oxazophorins, aziridines or imine-ethylenes, anti-metabolites,anti-tumor antibiotics, mitotic inhibitors, chromatin functioninhibitors, anti-angiogenesis agents, anti-estrogens, anti-androgens,chelating agents, Iron absorption stimulant, Cyclooxygenase inhibitors,Phosphodiesterase inhibitors, DNA inhibitors, DNA synthetis inhibitors,Apopstotis stimulants, Thymidylate inhibitors, T cell inhibitors,Interferon agonists, Ribonucleoside triphosphate reductase inhibitors,Aromatase inhibitors, Estrogen receptor antagonists, Tyrosine kinaseinhibitors, Cell cycle inhibitors, Taxane, Tubulin inhibitors,angiogenesis inhibitors, macrophage stimulants, Neurokinin receptorantagonists, Cannabinoid receptor agonists, Dopamine receptor agonsists,granulocytes stimulating factor agonists, Erythropoietin receptoragonists, somatostatin receptor agonists, LHRH agonists, Calciumsensitizers, VEGF receptor antagonists, interleukin receptorantagonists, osteoclast inhibitors, radical formation stimulants,endothelin receptor antagonists, Vinca alkaloid, anti-hormone orimmunomodulators or any other new drug that fullfills the activitycriteria of a cytotoxic or a toxin.

Such drugs are, for example, cited in the VIDAL 2010, on the pagedevoted to the compounds attached to the cancerology and hematologycolumn “Cytotoxics”, these cytotoxic compounds cited with reference tothis document are cited here as preferred cytotoxic agents.

More particularly, without limitation, the following drugs are preferredaccording to the invention: mechlorethamine, chlorambucol, melphalen,chlorydrate, pipobromen, prednimustin, disodic-phosphate, estramustine,cyclophosphamide, altretamine, trofosfamide, sulfofosfamide, ifosfamide,thiotepa, triethylenamine, altetramine, carmustine, streptozocin,fotemustin, lomustine, busulfan, treosulfan, improsulfan, dacarbazine,cis-platinum, oxaliplatin, lobaplatin, heptaplatin, miriplatin hydrate,carboplatin, methotrexate, pemetrexed, 5-fluoruracil, floxuridine,5-fluorodeoxyuridine, capecitabine, cytarabine, fludarabine, cytosinearabinoside, 6-mercaptopurine (6-MP), nelarabine, 6-thioguanine (6-TG),chlorodesoxyadenosine, 5-azacytidine, gemcitabine, cladribine,deoxycoformycin, tegafur, pentostatin, doxorubicin, daunorubicin,idarubicin, valrubicin, mitoxantrone, dactinomycin, mithramycin,plicamycin, mitomycin C, bleomycin, procarbazine, paclitaxel, docetaxel,vinblastine, vincristine, vindesine, vinorelbine, topotecan, irinotecan,etoposide, valrubicin, amrubicin hydrochloride, pirarubicin, elliptiniumacetate, zorubicin, epirubicin, idarubicin and teniposide, razoxin,marimastat, batimastat, prinomastat, tanomastat, ilomastat, CGS-27023A,halofuginon, COL-3, neovastat, thalidomide, CDC 501, DMXAA, L-651582,squalamine, endostatin, SU5416, SU6668, interferon-alpha, EMD121974,interleukin-12, IM862, angiostatin, tamoxifen, toremifene, raloxifene,droloxifene, iodoxyfene, anastrozole, letrozole, exemestane, flutamide,nilutamide, sprironolactone, cyproterone acetate, finasteride,cimitidine, bortezomid, Velcade, bicalutamide, cyproterone, flutamide,fulvestran, exemestane, dasatinib, erlotinib, gefitinib, imatinib,lapatinib, nilotinib, sorafenib, sunitinib, retinoid, rexinoid,methoxsalene, methylaminolevulinate, aldesleukine, OCT-43, denileukindiflitox, interleukin-2, tasonermine, lentinan, sizofilan, roquinimex,pidotimod, pegademase, thymopentine, poly I:C, procodazol, Tic BCG,corynebacterium parvum, NOV-002, ukrain, levamisole, 1311-chTNT, H-101,celmoleukin, interferon alfa2a, interferon alfa2b, interferon gammal a,interleukin-2, mobenakin, Rexin-G, teceleukin, aclarubicin, actinomycin,arglabin, asparaginase, carzinophilin, chromomycin, daunomycin,leucovorin, masoprocol, neocarzinostatin, peplomycin, sarkomycin,solamargine, trabectedin, streptozocin, testosterone, kunecatechins,sinecatechins, alitretinoin, belotecan hydrocholoride, calusterone,dromostanolone, elliptinium acetate, ethinyl estradiol, etoposide,fluoxymesterone, formestane, fosfetrol, goserelin acetate, hexylaminolevulinate, histrelin, hydroxyprogesterone, ixabepilone,leuprolide, medroxyprogesterone acetate, megesterol acetate,methylprednisolone, methyltestosterone, miltefosine, mitobronitol,nadrolone phenylpropionate, norethindrone acetate, prednisolone,prednisone, temsirrolimus, testolactone, triamconolone, triptorelin,vapreotide acetate, zinostatin stimalamer, amsacrine, arsenic trioxide,bisantrene hydrochloride, chlorambucil, chlortrianisene,cis-diamminedichloroplatinium, cyclophosphamide, diethylstilbestrol,hexamethylmelamine, hydroxyurea, lenalidomide, lonidamine,mechlorethanamine, mitotane, nedaplatin, nimustine hydrochloride,pamidronate, pipobroman, porfimer sodium, ranimustine, razoxane,semustine, sobuzoxane, mesylate, triethylenemelamine, zoledronic acid,camostat mesylate, fadrozole HCl, nafoxidine, aminoglutethimide,carmofur, clofarabine, cytosine arabinoside, decitabine, doxifluridine,enocitabine, fludarabne phosphate, fluorouracil, ftorafur, uracilmustard, abarelix, bexarotene, raltiterxed, tamibarotene, temozolomide,vorinostat, megastrol, clodronate disodium, levamisole, ferumoxytol,iron isomaltoside, celecoxib, ibudilast, bendamustine, altretamine,mitolactol, temsirolimus, pralatrexate, TS-1, decitabine, bicalutamide,flutamide, letrozole, clodronate disodium, degarelix, toremifenecitrate, histamine dihydrochloride, DW-166HC, nitracrine, decitabine,irinoteacn hydrochloride, amsacrine, romidepsin, tretinoin, cabazitaxel,vandetanib, lenalidomide, ibandronic acid, miltefosine, vitespen,mifamurtide, nadroparin, granisetron, ondansetron, tropisetron,alizapride, ramosetron, dolasetron mesilate, fosaprepitant dimeglumine,nabilone, aprepitant, dronabinol, TY-10721, lisuride hydrogen maleate,epiceram, defibrotide, dabigatran etexilate, filgrastim, pegfilgrastim,reditux, epoetin, molgramostim, oprelvekin, sipuleucel-T, M-Vax, acetylL-carnitine, donepezil hydrochloride, 5-aminolevulinic acid, methylaminolevulinate, cetrorelix acetate, icodextrin, leuprorelin,metbylphenidate, octreotide, amlexanox, plerixafor, menatetrenone,anethole dithiolethione, doxercalciferol, cinacalcet hydrochloride,alefacept, romiplostim, thymoglobulin, thymalfasin, ubenimex, imiquimod,everolimus, sirolimus, H-101, lasofoxifene, trilostane, incadronate,gangliosides, pegaptanib octasodium, vertoporfin, minodronic acid,zoledronic acid, gallium nitrate, alendronate sodium, etidronatedisodium, disodium pamidronate, dutasteride, sodium stibogluconate,armodafinil, dexrazoxane, amifostine, WF-10, temoporfin, darbepoetinalfa, ancestim, sargramostim, palifermin, R-744, nepidermin, oprelvekin,denileukin diftitox, crisantaspase, buserelin, deslorelin, lanreotide,octreotide, pilocarpine, bosentan, calicheamicin, maytansinoids andciclonicate.

For more detail, the person skilled in the art could refer to the manualedited by the “Association Francaise des Enseignants de ChimieThérapeutique” and entitled “traité de chimie thérapeutique, vol. 6,Médicaments antitumoraux et perspectives dans le traitement des cancers,edition TEC & DOC, 2003”.

In a second preferred embodiment of the invention, the immunoconjugateconsists of a binding protein linked to at least a radioisotope. Such animmunoconjugate is referred as an antibody-radioisotope conjugate (or“ARC”) when the binding protein is an antibody, or an antigen bindingfragment thereof.

For selective destruction of the tumor, the antibody may comprise ahighly radioactive atom. A variety of radioactive isotopes are availablefor the production of ARC such as, without limitation, At²¹¹, C¹³, N¹⁵,O¹⁷, Fl¹⁹, I¹²³, I¹³¹, I¹²⁵, In¹¹¹, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, tc⁹⁹ m,Bi²¹², P³², Pb²¹², radioactive isotopes of Lu, gadolinium, manganese oriron.

Any methods or processes known by the person skilled in the art can beused to incorporate such radioisotope in the ARC (see, for example“Monoclonal Antibodies in Immunoscintigraphy”, Chatal, CRC Press 1989).As non limitative example, tc⁹⁹ m or I¹²³, Re¹⁸⁶, Re¹⁸⁸ and In¹¹¹ can beattached via a cysteine residue. Y⁹⁰ can be attached via a lysineresidue. I¹²³ can be attached using the IODOGEN method (Fraker et al(1978) Biochem. Biophys. Res. Commun. 80: 49-57).

Several examples can be mentioned to illustrate the knowledge of theperson skilled in the art in the field of ARC such as Zevalin® which isan ARC composed of an anti-CD20 monoclonal antibody and In¹¹¹ or Y⁹⁰radioisotope bound by a thiourea linker-chelator (Wiseman et at (2000)Eur. Jour. Nucl. Med. 27(7):766-77; Wiseman et al (2002) Blood99(12):4336-42; Witzig et at (2002) J. Clin. Oncol. 20(10):2453-63;Witzig et al (2002) J. Clin. Oncol. 20(15):3262-69); or Mylotarg® whichis composed of an anti-CD33 antibody linked to calicheamicin, (U.S. Pat.Nos. 4,970,198; 5,079,233; 5,585,089; 5,606,040; 5,693,762; 5,739,116;5,767,285; 5,773,001). More recently, it can also be mentioned the ADCreferred as Adcetris (corresponding to the Brentuximab vedotin) whichhas been recently accepted by the FDA in the treatment of Hodgkin'slymphoma (Nature, vol. 476, pp 380-381, 25 Aug. 2011).

In a third preferred embodiment of the invention, the immunoconjugateconsists of a binding protein linked to at least a toxin. Such animmunoconjugate is referred as an antibody-toxin conjugate (or “ATC”)when the binding protein is an antibody, or an antigen binding fragmentthereof.

Toxins are effective and specific poisons produced by living organisms.They usually consist of an amino acid chain which can vary in molecularweight between a couple of hundred (peptides) and one hundred thousand(proteins). They may also be low-molecular organic compounds. Toxins areproduced by numerous organisms, e.g., bacteria, fungi, algae and plants.Many of them are extremely poisonous, with a toxicity that is severalorders of magnitude greater than the nerve agents.

Toxins used in ATC can include, without limitation, all kind of toxinswhich may exert their cytotoxic effects by mechanisms including tubulinbinding, DNA binding, or topoisomerase inhibition.

Enzymatically active toxins and fragments thereof that can be usedinclude diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin, and the tricothecenes.

Small molecule toxins, such as dolastatins, auristatins, atrichothecene, and CC1065, and the derivatives of these toxins that havetoxin activity, are also contemplated herein. Dolastatins andauristatins have been shown to interfere with microtubule dynamics, GTPhydrolysis, and nuclear and cellular division and have anticancer andantifungal activity.

“Linker”, “Linker Unit”, or “link” means a chemical moiety comprising acovalent bond or a chain of atoms that covalently attaches a bindingprotein to at least one cytotoxic agent.

Linkers may be made using a variety of bifunctional protein couplingagents such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). Carbon-14-labeled1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid(MX-DTPA) is an exemplary chelating agent for conjugation of cyctotoxicagents to the addressing system. Other cross-linker reagents may beBMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB,SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB,sulfo-SMCC, and sulfo-SMPB, and SVSB(succinimidyl-(4-vinylsulfone)benzoate) which are commercially available(e.g., from Pierce Biotechnology, Inc., Rockford, Ill., U.S.A).

The linker may be a “non cleavable” or “cleavable”.

In a preferred embodiment, it consists in a “cleavable linker”facilitating release of the cytotoxic agent in the cell. For example, anacid-labile linker, peptidase-sensitive linker, photolabile linker,dimethyl linker or disulfide-containing linker may be used. The linkeris, in a preferred embodiment, cleavable under intracellular conditions,such that cleavage of the linker releases the cytotoxic agent from thebinding protein in the intracellular environment.

For example, in some embodiments, the linker is cleavable by a cleavingagent that is present in the intracellular environment (e.g., within alysosome or endosome or caveolea). The linker can be, for example, apeptidyl linker that is cleaved by an intracellular peptidase orprotease enzyme, including, but not limited to, a lysosomal or endosomalprotease. Typically, the peptidyl linker is at least two amino acidslong or at least three amino acids long. Cleaving agents can includecathepsins B and D and plasmin, all of which are known to hydrolyzedipeptide drug derivatives resulting in the release of active druginside target cells. For example, a peptidyl linker that is cleavable bythe thiol-dependent protease cathepsin-B, which is highly expressed incancerous tissue, can be used (e.g., a Phe-Leu or a Gly-Phe-Leu-Glylinker). In specific embodiments, the peptidyl linker cleavable by anintracellular protease is a Val-Cit linker or a Phe-Lys linker. Oneadvantage of using intracellular proteolytic release of the cytotoxicagent is that the agent is typically attenuated when conjugated and theserum stabilities of the conjugates are typically high.

In other embodiments, the cleavable linker is pH-sensitive, i.e.,sensitive to hydrolysis at certain pH values. Typically, thepH-sensitive linker is hydrolyzable under acidic conditions. Forexample, an acid-labile linker that is hydrolyzable in the lysosome(e.g., a hydrazone, semicarbazone, thiosemicarbazone, cis-aconiticamide, orthoester, acetal, ketal, or the like) can be used. Such linkersare relatively stable under neutral pH conditions, such as those in theblood, but are unstable at below pH 5.5 or 5.0, the approximate pH ofthe lysosome. In certain embodiments, the hydrolyzable linker is athioether linker (such as, e.g., a thioether attached to the therapeuticagent via an acylhydrazone bond.

In yet other embodiments, the linker is cleavable under reducingconditions (e.g., a disulfide linker). A variety of disulfide linkersare known in the art, including, for example, those that can be formedusing SATA (N-succinimidyl-S-acetylthioacetate), SPDP(N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB(N-succinimidyl-3-(2-pyridyldithio)butyrate) and SMPT(N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene)-,SPDB and SMPT.

As non limitative example of non-cleavable or “non reductible” linkers,it can be mentioned the immunoconjugate Trastuzumab-DM1 (TDM1) whichcombines trastuzumab with a linked chemotherapy agent, maytansine(Cancer Research 2008; 68: (22). Nov. 15, 2008).

In a preferred embodiment, the immunoconjugate of the invention may beprepared by any method known by the person skilled in the art such as,without limitation, i) reaction of a nucleophilic group of the antigenbinding protein with a bivalent linker reagent followed by reaction withthe cytotoxic agent or ii) reaction of a nucleophilic group of acytotoxic agent with a bivalent linker reagent followed by reaction withthe nucleophilic group of the antigen binding protein.

Nucleophilic groups on antigen binding protein include, withoutlimitation, N-terminal amine groups, side chain amine groups, e.g.lysine, side chain thiol groups, and sugar hydroxyl or amino groups whenthe antigen binding protein is glycosylated. Amine, thiol, and hydroxylgroups are nucleophilic and capable of reacting to form covalent bondswith electrophilic groups on linker moieties and linker reagentsincluding, without limitation, active esters such as NHS esters, HOBtesters, haloformates, and acid halides; alkyl and benzyl halides such ashaloacetamides; aldehydes, ketones, carboxyl, and maleimide groups. Theantigen binding protein may have reducible interchain disulfides, i.e.cysteine bridges. The antigen binding proteins may be made reactive forconjugation with linker reagents by treatment with a reducing agent suchas DTT (dithiothreitol). Each cysteine bridge will thus form,theoretically, two reactive thiol nucleophiles. Additional nucleophilicgroups can be introduced into the antigen binding protein through anyreaction known by the person skilled in the art. As non limitativeexample, reactive thiol groups may be introduced into the antigenbinding protein by introducing one or more cysteine residues.

Immunoconjugates may also be produced by modification of the antigenbinding protein to introduce electrophilic moieties, which can reactwith nucleophilic substituents on the linker reagent or cytotoxic agent.The sugars of glycosylated antigen binding protein may be oxidized toform aldehyde or ketone groups which may react with the amine group oflinker reagents or cytotoxic agent. The resulting imine Schiff basegroups may form a stable linkage, or may be reduced to form stable aminelinkages. In one embodiment, reaction of the carbohydrate portion of aglycosylated antigen binding protein with either galactose oxidase orsodium meta-periodate may yield carbonyl (aldehyde and ketone) groups inthe protein that can react with appropriate groups on the drug. Inanother embodiment, proteins containing N-terminal serine or threonineresidues can react with sodium meta-periodate, resulting in productionof an aldehyde in place of the first amino acid.

In certain preferred embodiments, the linker unit may have the followinggeneral formula:-Ta-Ww-Yy-

wherein:

-T- is a stretcher unit;

a is 0 or 1;

-W- is an amino acid unit;

w is independently an integer ranging from 1 to 12;

-Y- is a spacer unit;

y is 0, 1 or 2.

The stretcher unit (-T-), when present, links the antigen bindingprotein to an amino acid unit (-W-). Useful functional groups that canbe present on the antigen binding protein, either naturally or viachemical manipulation, include sulfhydryl, amino, hydroxyl, the anomerichydroxyl group of a carbohydrate, and carboxyl. Suitable functionalgroups are sulfhydryl and amino. Sulfhydryl groups can be generated byreduction of the intramolecular disulfide bonds of the antigen bindingprotein, if present. Alternatively, sulhydryl groups can be generated byreaction of an amino group of a lysine moiety of the antigen bindingprotein with 2-iminothiolane or other sulfhydryl generating reagents. Inspecific embodiments, the antigen binding protein is a recombinantantibody and is engineered to carry one or more lysines. Morepreferably, the antigen binding protein can be engineered to carry oneor more Cysteines (cf. ThioMabs).

In certain specific embodiments, the stretcher unit forms a bond with asulfur atom of the antigen binding protein. The sulfur atom can bederived from a sulfhydryl (—SH) group of a reduced antigen bindingprotein.

In certain other specific embodiments, the stretcher unit is linked tothe antigen binding protein via a disulfide bond between a sulfur atomof the antigen binding protein and a sulfur atom of the stretcher unit.

In other specific embodiments, the reactive group of the stretchercontains a reactive site that can be reactive to an amino group of theantigen binding protein. The amino group can be that of an arginine or alysine. Suitable amine reactive sites include, but are not limited to,activated esters such as succinimide esters, 4-nitrophenyl esters,pentafluorophenyl esters, anhydrides, acid chlorides, sulfonylchlorides, isocyanates and isothiocyanates.

In yet another aspect, the reactive function of the stretcher contains areactive site that is reactive to a modified carbohydrate group that canbe present on the antigen binding protein. In a specific embodiment, theantigen binding protein is glycosylated enzymatically to provide acarbohydrate moiety (to be noticed that, when the antigen bindingprotein is an antibody, said antibody is generally naturallyglycosylated). The carbohydrate may be mildly oxidized with a reagentsuch as sodium periodate and the resulting carbonyl unit of the oxidizedcarbohydrate can be condensed with a stretcher that contains afunctionality such as a hydrazide, an oxime, a reactive amine, ahydrazine, a thiosemicarbazide, a hydrazine carboxylate, or anarylhydrazide.

The amino acid unit (-W-) links the stretcher unit (-T-) to the Spacerunit (-Y-) if the spacer unit is present, and links the stretcher unitto the cytotoxic agent if the spacer unit is absent.

As above mentioned, -Ww- may be a dipeptide, tripeptide, tetrapeptide,pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide,decapeptide, undecapeptide or dodecapeptide unit

In some embodiments, the amino acid unit may comprise amino acidresidues such as, without limitation, alanine, valine, leucine,isoleucine, methionine, phenylalanine, tryptophan, proline, lysineprotected with acetyl or formyl, arginine, arginine protected with tosylor nitro groups, histidine, ornithine, ornithine protected with acetylor formyl and citrulline. Exemplary amino acid linker components includepreferably a dipeptide, a tripeptide, a tetrapeptide or a pentapeptide.

Exemplary dipeptides include: Val-Cit, Ala-Val, Lys-Lys, Cit-Cit,Val-Lys, Ala-Phe, Phe-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Trp-Cit,Phe-Ala, Phe-N⁹-tosyl-Arg, Phe-N⁹-Nitro-Arg.

Exemplary tripeptides include: Val-Ala-Val, Ala-Asn-Val, Val-Leu-Lys,Ala-Ala-Asn, Phe-Phe-Lys, Gly-Gly-Gly, D-Phe-Phe-Lys, Gly-Phe-Lys.

Exemplary tetrapeptide include: Gly-Phe-Leu-Gly (SEQ ID NO. 33),Ala-Leu-Ala-Leu (SEQ ID NO. 34).

Exemplary pentapeptide include: Pro-Val-Gly-Val-Val (SEQ ID NO. 35).

Amino acid residues which comprise an amino acid linker componentinclude those occurring naturally, as well as minor amino acids andnon-naturally occurring amino acid analogs, such as citrulline. Aminoacid linker components can be designed and optimized in theirselectivity for enzymatic cleavage by a particular enzyme, for example,a tumor-associated protease, cathepsin B, C and D, or a plasminprotease.

The amino acid unit of the linker can be enzymatically cleaved by anenzyme including, but not limited to, a tumor-associated protease toliberate the cytotoxic agent.

The amino acid unit can be designed and optimized in its selectivity forenzymatic cleavage by a particular tumor-associated protease. Thesuitable units are those whose cleavage is catalyzed by the proteases,cathepsin B, C and D, and plasmin.

The spacer unit (-Y-), when present, links an amino acid unit to thecytotoxic agent. Spacer units are of two general types: self-immolativeand non self-immolative. A non self-immolative spacer unit is one inwhich part or all of the spacer unit remains bound to the cytotoxicagent after enzymatic cleavage of an amino acid unit from theimmunoconjugate. Examples of a non self-immolative spacer unit include,but are not limited to a (glycine-glycine) spacer unit and a glycinespacer unit. To liberate the cytotoxic agent, an independent hydrolysisreaction should take place within the target cell to cleave theglycine-drug unit bond.

In another embodiment, a non self-immolative the spacer unit (-Y-) is-Gly-.

In one embodiment, the immunoconjugate lacks a spacer unit (y=0).Alternatively, an imunoconjugate containing a self-immolative spacerunit can release the cytotoxic agent without the need for a separatehydrolysis step. In these embodiments, -Y- is a p-aminobenzyl alcohol(PAB) unit that is linked to -Ww- via the nitrogen atom of the PABgroup, and connected directly to -D via a carbonate, carbamate or ethergroup.

Other examples of self-immolative spacers include, but are not limitedto, aromatic compounds that are electronically equivalent to the PABgroup such as 2-aminoimidazol-5-methanol derivatives and ortho orpara-aminobenzylacetals. Spacers can be used that undergo facilecyclization upon amide bond hydrolysis, such as substituted andunsubstituted 4-aminobutyric acid amides, appropriately substitutedbicyclo[2.2.1] and bicyclo[2.2.2] ring systems and2-aminophenylpropionic acid amides.

In an alternate embodiment, the spacer unit is a branchedbis(hydroxymethyl)styrene (BHMS) unit, which can be used to incorporateadditional cytotoxic agents.

An embodiment of the invention is an immunoconjugate comprising anantigen binding protein which i) specifically binds to the human proteinAxl, preferably having the sequence SEQ ID NO. 29 or 30 or naturalvariant sequence thereof, and ii) is internalized following its bindingto said human protein Axl, said antigen binding protein being conjugatedto at least one drug, toxin or radioisotope.

An embodiment of the invention relates to an immunoconjugate havingformula (I)Ab-(L-D)_(n)  (I)

or a pharmaceutically acceptable salt or solvate thereof, wherein:

Ab is an antigen binding protein which i) specifically binds to theprotein Axl, and ii) is internalized into a mammalian cell following itsbinding to said protein Axl when said Axl protein is expressed at thesurface of said mammalian cell;

L is a linker;

D is a drug, a toxin and/or a radioisotope;

n is 1 to 12.

In a particular embodiment, the immunoconjugate of the invention has theformula (I)Ab-(L-D)_(n)  (I)

or a pharmaceutically acceptable salt or solvate thereof, wherein:

Ab is an antigen binding protein which i) specifically binds to theprotein Axl with an EC₅₀ of at least 10⁻⁹ M, preferentially between 10⁻⁹and 10⁻¹² M., and ii) is internalized into a mammalian cell followingits binding to said protein Axl when said Axl protein is expressed atthe surface of said mammalian cell;

L is a linker;

D is a drug, a toxin and/or a radioisotope;

n is 1 to 12.

In another particular embodiment, the immunoconjugate of the inventionhas the formula (I)Ab-(L-D)_(n)  (I)

or a pharmaceutically acceptable salt or solvate thereof, wherein:

Ab is an antigen binding protein which i) specifically binds to theprotein Axl, and ii) induces a reduction of MFI of at least 200;

L is a linker;

D is a drug, a toxin and/or a radioisotope;

n is 1 to 12.

In a particular embodiment, the immunoconjugate of the invention has theformula (I)Ab-(L-D)_(n)  (I)

or a pharmaceutically acceptable salt or solvate thereof, wherein:

Ab is an antigen binding protein which i) specifically binds to theprotein Axl with an EC₅₀ of at least 10⁻⁹ M, preferentially between 10⁻⁹and 10⁻¹² M., and ii) induces a reduction of MFI of at least 200;

L is a linker;

D is a drug, a toxin and/or a radioisotope;

n is 1 to 12.

In an embodiment of the invention, the antigen binding protein of theimmunoconjugate of the present invention is selected by the method forthe screening of a compound, or a binding fragment thereof, capable ofdelivering or internalizing a molecule of interest into mammalian cellsaccording to the present invention

In an embodiment of the invention, the antigen binding protein induces areduction of MFI of at least 200 as measured on SN12C cells.

In an embodiment of the invention, the antigen binding protein binds tothe protein Axl with an EC₅₀ of at least 10⁻⁹ M, preferentially between10⁻⁹ and 10⁻¹² M, as measured on SN12C cells.

For the avoidance of doubt, the linker L can be selected through anylinkers known by the person skilled in the art. As non limitativeexample, the linker L is selected in the group of linkers describedabove in the present specification.

For the avoidance of doubt, the Drug D can be selected through any drug,a toxin and/or a radioisotope known by the person skilled in the art. Asnon limitative example, the Drug D is selected in the group of drugs,toxins or radioisotopes described above in the present specification.

Finally, the invention relates to an immunoconjugate as above describedfor use in the treatment of cancer.

Cancers can be preferably selected through Axl-related cancers includingtumoral cells expressing or over-expressing whole or part of the proteinAxl at their surface.

More particularly, said cancers are breast, colon, esophageal carcinoma,hepatocellular, gastric, glioma, lung, melanoma, osteosarcoma, ovarian,prostate, rhabdomyosarcoma, renal, thyroid, uterine endometrial cancer,mesothelioma, oral squamous carcinoma and any drug resistance phenomena.Another object of the invention is a pharmaceutical compositioncomprising the immunoconjugate as described in the specification.

More particularly, the invention relates to a pharmaceutical compositioncomprising the immunoconjugate of the invention with at least anexcipient and/or a pharmaceutical acceptable vehicle.

In the present description, the expression “pharmaceutically acceptablevehicle” or “excipient” is intended to indicate a compound or acombination of compounds entering into a pharmaceutical composition notprovoking secondary reactions and which allows, for example,facilitation of the administration of the active compound(s), anincrease in its lifespan and/or in its efficacy in the body, an increasein its solubility in solution or else an improvement in itsconservation. These pharmaceutically acceptable vehicles and excipientsare well known and will be adapted by the person skilled in the art as afunction of the nature and of the mode of administration of the activecompound(s) chosen.

Preferably, these immunoconjugates will be administered by the systemicroute, in particular by the intravenous route, by the intramuscular,intradermal, intraperitoneal or subcutaneous route, or by the oralroute. In a more preferred manner, the composition comprising theimmunoconjugates according to the invention will be administered severaltimes, in a sequential manner.

Their modes of administration, dosages and optimum pharmaceutical formscan be determined according to the criteria generally taken into accountin the establishment of a treatment adapted to a patient such as, forexample, the age or the body weight of the patient, the seriousness ofhis/her general condition, the tolerance to the treatment and thesecondary effects noted.

Other characteristics and advantages of the invention appear in thecontinuation of the description with the examples and the figures whoselegends are represented below.

FIGURE LEGENDS

FIG. 1: in vitro cytotoxicity assay using Mab-zap conjugated secondaryantibody on SN12C cells.

FIG. 2A, FIG. 2B and FIG. 2C: Binding specificity of 1613F12 on theimmobilized rhAxl-Fc protein (FIG. 2A), rhDtk-Fc (FIG. 2B) or rhMer-Fc(FIG. 2C) proteins by ELISA.

FIG. 3: FACS analysis of the 1613F12 binding on human tumor cells

FIG. 4: ELISA on the immobilized rmAxl-Fc protein (“rm” for murinerecombinant).

FIG. 5: 1613F12 binding on COS7 cells as determined by indirectlabelling protocol using flow cytometry method.

FIG. 6: Competition ELISA of Gas6 binding using 1613F12.

FIG. 7: Epitope binding analysis by western Blot using SN12C celllysate. NH (no heat); NR (no reduction); H (heat); R (reduction). GAPDHdetection attests to the correct sample loading on the gel.

FIG. 8A and FIG. 8B: Study of Axl downregulation after 1613F12 bindingon SN12C cells by Western Blot with FIG. 8A—Western blot imagerepresentative of the 3 independent experiments performed (The westernblot analysis was performed after a 4 h and 24 h incubation of the1613F12 on SN12C cells); and FIG. 8B—Optical density quantification ofthe presented film using “QuantityOne” software.

FIG. 9A, FIG. 9B and FIG. 9C: Immunofluorescence microscopy of SN12Ccells after incubation with the 1613F12 FIG. 9A—Photographs of the mIgG1isotype control conditions both for the membrane and the intracellularstaining. FIG. 9B—Membrane staining. FIG. 9C—Intracellular staining ofboth Axl receptor using the 1613F12 and of the early endosome markerEEA1. Image overlays are presented bellow and co-localizationsvisualized are indicated by the arrows.

FIG. 10: Effect of 1613F12 on in vitro SN12C cells proliferationcompared to the effect of the mIgG1 isotype control antibody.

FIG. 11A through FIG. 11K: Direct cytotoxicity assays of the1613F12-saporin immunoconjugate using various human tumor cell lines.FIG. 11A—SN12C, FIG. 11B—Calu-1, FIG. 11C—A172, FIG. 11D—A431, FIG.11E—DU145, FIG. 11F—MDA-MB435S, FIG. 11G—MDA-MB231, FIG. 11H—PC3, FIG.11I—-NCI-H226, FIG. 11J—NCI-H125, FIG. 11K—Panc1.

FIG. 12A, FIG. 12B, FIG. 12C: Antibody binding competition between FIG.12A 427B7, FIG. 12B 11D12 and FIG. 12C 320G10 obtained by Biacore.

FIG. 13: Direct cytotoxicity assays using SN12C cells (in presence of1A4-saporin immunoconjugate and an isotype control coupled to saporin).

EXAMPLES Example 1: Axl Receptor Internalization

As an immunoconjugate approach is more efficient when the targetedantigen is an internalizing protein, Axl receptor internalization usingMab-Zap cytotoxicity assay on human tumor cell lines was studied. Moreprecisely, the Mab-Zap reagent is a chemical conjugate including anaffinity purified goat anti-mouse IgG and the ribosome-inactivatingprotein, saporin. If internalization of the immune complex occurs,saporin breaks away from the targeting agent and inactivates theribosomes, resulting in protein synthesis inhibition and, ultimately,cell death. Cell viability determination after 72 hours of incubationwith the anti-Axl antibody or with mIgG1 isotype control antibody onAxl-positive cells allows concluding on the anti-Axl antibody-inducedAxl receptor internalization. For this study, several other murineanti-Axl antibodies were tested.

For this example highly Axl-positive cells, as determined using Qifikitreagent (Dako), were used. Data are presented in the following table 5.

TABLE 5 Antigen binding capacity of the MAB154 anti-Axl commercialantibody determined for the human renal cancer SN12C cells RTK AXL Cellline Antibody MAB154 SN12C >100 000

In the following example, the SN12C cells were used as non limitativeexample. Any other cell line expressing appropriate level of Axlreceptor on its cell surface could be used.

Concentration ranges of either the 1613F12 or any other tested murineanti-Axl antibody or the mIgG1 isotype control antibody werepre-incubated with 100 ng of Mab-Zap (Advanced targeting systems)secondary antibody in cell culture medium for 30 min at RT. Thesemixtures were loaded on sub-confluent SN12C cells plated in white96-well plate microplate. Plates were incubated for 72 h at 37° C. inpresence of 5% CO₂. Cell viability was determined using a Cell Titer Glocell proliferation method according to the manufacturer's instructions(Promega). Several controls are performed: i) without any secondaryimmunoconjugate and ii) without primary antibody. In parallel, assaysare performed with a mIgG1 isotype control.

Data obtained with the 1613F12 antibody are presented in the FIG. 1 andthe data obtained with the other murine anti-Axl antibodies arepresented in Table 6. The 1613F12 shows a maximal cytotoxic effect onthe SN12C cells of ˜36%. The percentages of maximum cytotoxicityobtained with other anti-Axl antibodies are listed in Table 6.

No cytotoxic effect was observed in presence of the 9G4 antibody,considered as mIgG1 isotype control in the experiment. No cytotoxicitywas observed in wells containing only primary antibodies (data notshown).

Using different anti-Axl antibodies, maximal cytotoxicity percentagesare comprised between 36.3% and 69% after 3 days of incubation with theantibodies.

Referring to the obtained cytotoxicity effect in this experiment, theAxl receptor appears to be a convenient antigen to target for animmunoconjugate approach as the various immune complexes comprisingAxl-Axl anti-antibody-MabZap can trigger an effective cytotoxicity ofthe targeted cells.

Thus the Axl receptor appears to be a convenient antigen to target foran immunoconjugate approach as the immune complex comprisingAxl-1613F12-MabZap, or all other anti-Axl antibodies of the invention,triggers an effective cytotoxicity of the targeted cells.

TABLE 6 Percentages of the maximum cytotoxicity obtained in indirectSN12C cytotoxicity assays % cytotoxicity max 1A4 direct* 11D12 54.2%320G10 41.4% 427B7 36.3% 517F1 66.5% 530C8 50.2% 534G9 49.9% 535F7 55.6%547A1 69.0% 1023F10 49.7% 1614G5 65.1% *Axl antibody tested in direct invitro SN12C cytotoxicity assay

Example 2: Generation of an Antibody Against rhAxl-ECD

Various strategies were used to generate murine antibodies targeting thehuman Axl extracellular domain.

The detailed protocol used to obtain the 1613F12 antibody is describedbellow.

To generate murine monoclonal antibodies (Mabs) against humanextracellular domain (ECD) of the Axl receptor, 5 BALB/c mice wereimmunized 5-times s.c. with 15-20·10⁶ CHO-Axl cells and twice with 20 μgof the rh Axl-ECD. The first immunization was performed in presence ofComplete Freund Adjuvant (Sigma, St Louis, Md., USA). Incomplete Freundadjuvant (Sigma) was added for following immunizations.

Three days prior to the fusion, immunized mice were boosted with both20·10⁶ CHO-Axl cells and 20 μg of the rhAxl-ECD with IFA.

To generate hybridomas, splenocytes and lymphocytes were prepared byperfusion of the spleen and by mincing of the proximal lymph nodes,respectively, harvested from 1 out of the 5 immunized mice (selectedafter sera titration) and fused to SP2/0-Ag14 myeloma cells (ATCC,Rockville, Md., USA). The fusion protocol is described by Kohler andMilstein (Nature, 256:495-497, 1975). Fused cells are then subjected toHAT selection. In general, for the preparation of monoclonal antibodiesor their functional fragments, especially of murine origin, it ispossible to refer to techniques which are described in particular in themanual “Antibodies” (Harlow and Lane, Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory, Cold Spring Harbor N.Y., pp. 726, 1988).

Approximately 10 days after the fusion, colonies of hybrid cells werescreened. For the primary screen, supernatants of hybridomas wereevaluated for the secretion of Mabs raised against the Axl ECD proteinusing an ELISA. In parallel, a FACS analysis was performed to selectMabs able to bind to the cellular form of Axl present on the cellsurface of both wt CHO and Axl expressing CHO cells (ATCC).

As soon as possible, selected hybridomas were cloned by limit dilutionand subsequently screened for their reactivity against the Axl ECDprotein. Cloned Mabs were then isotyped using an Isotyping kit (cat#5300.05, Southern Biotech, Birmingham, Ala., USA). One clone obtainedfrom each hybridoma was selected and expanded. Once produced theanti-Axl antibodies were further studied for their ability to beinternalized following Axl binding on the cell-surface. In parallel, thehybridomas are deposited at the CNCM.

ELISA assays are performed as followed either using pure hybridomasupernatant or, when IgG content in supernatants was determined,titration was realized starting at 5 μg/ml. Then a ½ serial dilution wasperformed in the following 11 rows. Briefly, 96-well ELISA plates(Costar 3690, Corning, N.Y., USA) were coated 50 μl/well of the rhAxl-Fc protein (R and D Systems, cat No. 154-AL) or rhAxl-ECD at 2 μg/mlin PBS overnight at 4° C. The plates were then blocked with PBScontaining 0.5% gelatin (#22151, Serva Electrophoresis GmbH, Heidelberg,Germany) for 2 h at 37° C. Once the saturation buffer discarded byflicking plates, 50 μl of pure hybridoma cell supernatants or 50 μl of a5 μg/ml solution were added to the ELISA plates and incubated for 1 h at37° C. After three washes, 50 μl horseradish peroxidase-conjugatedpolyclonal goat anti-mouse IgG (#115-035-164, Jackson Immuno-ResearchLaboratories, Inc., West Grove, Pa., USA) was added at a 1/5000 dilutionin PBS containing 0.1% gelatin and 0.05% Tween 20 (w:w) for 1 h at 37°C. Then, ELISA plates were washed 3-times and the TMB (#UP664782,Uptima, Interchim, France) substrate was added. After a 10 minincubation time at room temperature, the reaction was stopped using 1 Msulfuric acid and the optical density at 450 nm was measured.

For the selection by flow cytometry, 10⁵ cells (CHO wt or CHO-Axl) wereplated in each well of a 96 well-plate in PBS containing 1% BSA and0.01% sodium azide (FACS buffer) at 4° C. After a 2 min centrifugationat 2000 rpm, the buffer was removed and hybridoma supernatants orpurified Mabs (1 μg/ml) to be tested were added. After 20 min ofincubation at 4° C., cells were washed twice and an Alexa 488-conjugatedgoat anti-mouse antibody 1/500° diluted in FACS buffer (#A11017,Molecular Probes Inc., Eugene, USA) was added and incubated for 20 minat 4° C. After a final wash with FACS buffer, cells were analyzed byFACS (Facscalibur, Becton-Dickinson) after addition of propidium iodideto each tube at a final concentration of 40 μg/ml. Wells containingcells alone and cells incubated with the secondary Alexa 488-conjugatedantibody were included as negative controls. Isotype controls were usedin each experiment (Sigma, ref M90351MG). At least 5000 cells wereassessed to calculate the mean value of fluorescence intensity (MFI).

More precisely, the fusion was performed with 300.10⁶ of harvestedsplenocytes and 300·10⁶ myeloma cells (1:1 ratio). Two hundred thousandcells of the resulting cell suspension were then plated at 2·10⁶ cell/mlin 30 96-well plates.

A first screen (around Day 14 after fusion) both by ELISA on therhAxl-ECD protein and by FACS analysis using the both wt CHO and Axlexpressing CHO cells allowed to select 10 hybridomas presenting opticaldensities (ODs) above 1 on the rh Axl-ECD coating and MFI bellow 50 onwt CHO cells and above 200 on CHO-Axl cells.

These 10 hybridomas were expanded and cloned by limit dilution. One96-well plate was prepared for each code. Nine days after plating,supernatants from cloning plates were first screened by ELISA for theirbinding specificity for the extracellular domain of the rh Axl-ECDprotein. Three clones of each code were expanded and isotyped. Onceproduced the anti-Axl antibodies were further studied for their abilityto be internalized following Axl binding on the cell-surface.

The protocols applied to give rise to the other anti-Axl antibodies aresummarized in Table 7.

TABLE 7 Immunization protocols Injections Immunogen Site of injectionOrgan source Hybridoma screen Antibody 3-5 immunizations + 5-20 μg of rhAxl protein s.c. spleen ELISA rhAxl 1A4, 320G10, 1 boost (i.v or i.p.)(monomeric or dimeric) proximal lymph FACS DU145 11D12, 517F1, 530C8,nodes 534G9, 535F7, 547A1, 427B7, 1023F10 5 immunizations 15-20 10⁶CHO-Axl cells s.c. spleen ELISA rh Axl 1613F12 2 immunization + 20 μgrhAxl protein proximal lymph FACS CHO Axl/CHO wt 1614G5 1 boost (i.v ori.p.) nodes

Moreover, the isotype of the anti-Axl antibodies are given in thefollowing Table 8.

In addition, the epitope diversity of the obtained Axl antibodies wasassessed by antibody binding competition. Epitope clustering experimentswere run out on a Biacore X device. This instrument based on the opticalphenomenon of surface plasmon resonance (SPR) enables the detection andmeasurement of protein-protein interactions in real time, without anylabelling.

Briefly, the experiments were realized using a sensor chip CM5 as thebiosensor. An anti-polyhistidine mouse IgG1 antibody (R and D Systems,MAB050) was coupled on both flow cells (FC1 and FC2) using aminecoupling chemistry.

The experiments were carried out at a flow rate of 10-20 μl/min in aHBS-EP buffer at 25° C. The recombinant hAxl-Fc chimera protein (R and DSystems, 154-AL-100) was injected during one minute at the concentrationof 5-10-20 μg/ml on both flowcells. Typically, this injection capturedbetween 300 to 500 RU of protein. A first antibody (culture supernatantor purified antibody at the concentration of 50 μg/ml) is injected onthe flowcell 1 during 1-2 min. in order to reach a saturation of thecaptured antigen. The second antibody was the injected on both flowcells(culture supernatant or purified antibody at the concentration of 50μg/ml) during 60 s. Both antibodies are classed in the same epitopicgroup when the signal of the second antibody on the flowcell 1 is atleast 10 times lower than the response on the flowcell 2.

At the end of each cycle, the surfaces are regenerated by injecting a 10mM glycine hydrochloride pH1.5 solution during 30 s.

The epitopic group of the anti-Axl antibodies is precised in Table 8.

Examples of Biacore data are reported in FIG. 12 which illustrates thecompetition between 427B7, 11D12 and 320G10. The same way of analysiswas applied for the other anti-Axl antibodies of the invention.

TABLE 8 Isotype and Epitopic group of the Axl antibodies. IsotypeEpitopic group CNCM 1A4 IgG1 K group #B I-3947 11D12 IgG1 K group #BI-4116 320G10 IgG1 K group #C I-4514 427B7 IgG1 K group #A I-4518 517F1IgG1 K group #B I-4502 530C8 IgG1 K group #B1 I-4728 534G9 IgG1 K group#B2 I-4729 535F7 IgG1 K group #B I-4510 547A1 IgG1 K group #B I-45061023F10 IgG1 K group #B I-4507 1613F12 IgG1 K group #B I-4505 1614G5IgG1 K group #A I-4519

They spread over 5 epitopic clusters.

Example 3: Axl Binding Specificity

In this example, the binding of the anti-Axl antibodies was firststudied on the rhAxl-Fc protein. Then, their binding on the two othermembers of the TAM family, rhDtk-Fc and rhMer-Fc, was studied.

Briefly, the recombinant human Axl-Fc (R and D systems, cat No.154AL/CF), rhDtk (R and D Systems, cat No. 859-DK) or rhMer-Fc (R and DSystems, cat No. 891-MR) proteins were coated overnight at 4° C. toImmulon II 96-well plates and, after a 1 h blocking step with a 0.5%gelatine solution, purified anti-Axl antibodies were added for anadditional 1 h at 37° C. at starting concentration of 5 μg/ml (3.3310⁻⁸M). Then ½ serial dilutions were done over 12 columns. Plates werewashed and a goat anti-mouse (Jackson) specific IgG-HRP was added for 1h at 37° C. Reaction development was performed using the TMB substratesolution. The commercial anti-Axl Mab 154 antibody was also used inparallel (data not shown). Coating controls were performed in presenceof a goat anti-human IgG Fc polyclonal serum labelled with HRP (Jackson,ref 109-035-098) and/or in presence of a HRP-coupled anti-Histidineantibody (R and D Systems, ref: MAB050H). No non specific binding wasobserved in absence of primary antibody (diluant). Results obtained withthe 1613F12 antibody are graphically represented in FIGS. 2A, 2B and 2C,respectively. Data obtained for the other anti-Axl antibodies arereported in the Table 9.

TABLE 9 Binding specificty of the Axl antibodies assessed by ELISA usinghuman recombinant proteins* rhAxl-Fc rhMer-Fc rhDtk-Fc 1A4 2.816 0.2980.142 11D12 2.994 0.281 0.104 320G10 2.737 0.287 0.107 427B7 2.840 0.2840.144 517F1 2.862 0.300 0.112 530C8 2.712 0.250 0.115 534G9 2.629 0.2260.108 535F7 2.721 0.235 0.115 547A1 2.691 0.227 0.099 1023F10 2.6760.247 0.114 1613F12 2.806 0.252 0.121 1614G5 2.592 0.275 0.122 *Valuesof ODs (450 nm) obtained in presence of a 5 μg/ml concentration of Axlantibody are reported here.

This example shows that the 1613F12 antibody only binds to the rhAxl-Fcprotein and does not bind on the two other members of the TAM family,rhDtk or rhMer. No cross-specificity of binding of the 1613F12 antibodyis observed between TAM members. Similar results are observed for allthe other tested anti-Axl antibodies. Indeed, they present a specificityof binding restricted to Axl; they do not recognize the other members ofthe TAM family.

Example 4: 1613F12 and Other Anti-Axl Antibodies Recognized the CellularForm of Axl Expressed on Tumor Cells

Cell surface Axl expression level on human tumor cells was firstestablished using a commercial Axl antibody (R and D Systems, ref:MAB154) in parallel of calibration beads to allow the quantification ofthe Axl expression level. Then, binding of the cell-surface Axl wasstudied using the anti-Axl antibodies of the invention. In both cases,the experimental conditions were as briefly described bellow.

For cell surface binding studies, two fold serial dilutions of a 10μg/ml (6.66 10⁻⁸ M) primary antibody solution (anti-Axl antibodies,MAB154 anti-Axl commercial antibody or mIgG1 isotype control 9G4 Mab)are prepared on 10 points and applied on 2.10⁵ cells for 20 min at 4° C.After 3 washes in phosphate-buffered saline (PBS) supplemented with 1%BSA and 0.01% NaN₃, cells were incubated with the secondary Goatanti-mouse Alexa 488 antibody (1/500° dilution) for 20 minutes at 4° C.After 3 additional washes in PBS supplemented with 1% BSA and 0.1% NaN₃,cells were analyzed by FACS (FACS, Becton-Dickinson). At least 5000cells were assessed to calculate the mean value of fluorescenceintensity.

For quantitative ABC determination using the MAB154 Axl antibody,QIFIKIT® calibration beads are used. In parallel with the QIFIKIT®beads, cells are incubated, with a Polyclonal Goat Anti-MouseImmunoglobulins/FITC (Goat F(ab′)₂) at a saturating concentration. Thenumber of antigenic sites on the studied cells is then determined byinterpolation of the calibration curve (the fluorescence intensity ofthe individual bead populations against the number of Mab molecules onthe beads).

4.1. Quantification of Cell-Surface Axl Expression Level

Axl expression level on the surface of human tumor cells was determinedby flow cytometry using indirect immunofluorescence assay (QIFIKIT®method (Dako, Denmark), a quantitative flow cytometry kit for assessingcell surface antigens. A comparison of the mean fluorescence intensity(MFI) of the known antigen levels of the beads via a calibration graphpermits determination of the antibody binding capacity (ABC) of the celllines.

Table 10 presents Axl expression level detected on the surface ofvarious human tumor cell lines (SN12C, Calu-1, A172, A431, DU145,MDA-MB435S, MDA-MB231, PC3, NCI-H226, NCI-H125, MCF7, Panc1) (ATCC, NCI)as determined using QIFIKIT® using the Axl commercial antibody MAB154 (Rand D Systems). Values are given as Antigen Binding Complex (ABC).

TABLE 10 MCF7 NCI-H125 MDA-MB-435S Panc1 MDA-MB-231 Calu-1 SN12C TumorBreast NSCLC Breast Pancreas Breast Lung Renal type/organ ABC 71 5 54017 814 36 809 61 186 >100 000 >100 000 (Qifikit) A172 A431 DU-145 PC3NCI-H226 Tumor glioblastoma Epidermoid Prostate prostate NSCLCtype/organ carcinoma ABC 52421 3953 55268 8421 32142 (Qifikit)

Results obtained with a commercial Axl monoclonal antibody (MAB154)showed that Axl receptor is expressed at various levels depending of theconsidered human tumor cell.

4.2. Axl Detection by 1613F12 and Anti-Axl Antibodies According to theInvention on Human Tumor Cells

Axl binding obtained using the 1613F12 was studied more extensively on apanel of different human tumor cells. Data are graphically presented inFIG. 3.

The binding of the other anti-Axl antibodies was documented using theSN12C human renal tumor cells only.

Anti-Axl antibody dose response curves were run. MFIs obtained with the1613F12 antibody using the various human tumor cells were then analysedwith Prism software. Data obtained with the 1613F12 are presented inFIG. 3. The EC₅₀ of the binding on SN12C cells obtained for the othertested anti-Axl antibodies are reported in Table 11.

TABLE 11 Binding of the Axl antibodies on SN12c cells studied by flowcytometry EC₅₀ SN12C binding (M) 1A4 1.E−09 11D12 2.E−10 320G10 4.E−10427B7 5.E−10 517F1 7.E−10 530C8 1.E−09 534G9 4.E−10 535F7 8.E−11 547A12.E−10 1023F10 1.E−10 1614G5 9.E−10

As illustrated in FIG. 3, data indicate that the 1613F12 bindsspecifically to the membrane Axl receptor as attested by the saturationcurve profiles. Similar profiles were obtained with the other testedantibodies (data not shown). The values of the EC₅₀ of anti-Axl antibodybinding on SN12C cells are comprised between 10⁻⁹M and 10⁻¹² M.

Example 5: 1613F12 Inter-Species Crosspecificity

To address the species cross-specificity of the 1613F12, two specieswere considered: mouse and monkey. First the binding on the recombinantmouse (rm) Axl receptor is studied by ELISA (FIG. 4). Then flowcytometry experiments were performed using monkey COS7 cells as thesecells express the Axl receptor on their surface (FIG. 5). The COS7 cellline was obtained by immortalizing a CV-1 cell line derived from kidneycells of the African green monkey with a version of the SV40 genome thatcan produce large T antigen but has a defect in genomic replication.

rmAxl-Fc ELISA

Briefly, the recombinant mouse Axl-Fc (R and D systems, cat No.854-AX/CF) proteins were coated overnight at 4° C. to Immulon II 96-wellplates and, after a 1 h blocking step with a 0.5% gelatine solution, the1613F12 purified antibody was added for one additional hour at 37° C. atstarting concentration of 5 μg/ml (3.33 10⁻⁸ M). Then ½ serial dilutionswere done over 12 columns. Plates were then washed and a goat anti-mouse(Jackson) specific IgG HRP was added for 1 h at 37° C. Reactiondevelopment was performed using the TMB substrate solution. Thecommercial mouse anti-Axl Mab 154 antibody is also used in parallel.Coating controls are performed in presence of a goat anti-human IgG Fcpolyclonal serum coupled with HRP (Jackson, ref 109-035-098) and/or inpresence of a HRP-coupled anti-Histidine antibody (R and D Systems, ref:MAB050H). No specific binding is observed in the absence of primaryantibody (diluant).

Results are represented in FIG. 4. This figure shows that the 1613F12Mab described in the present invention does not bind to the murine AxlECD domain.

FACS COS7

For 1613F12 cellular binding studies using COS7 cells, 2·10⁵ cells wereincubated with an antibody concentration range prepared by ½ serialdilution (12 points) of a 10 μg/ml (6.66 10⁻⁸ M) antibody solution of1613F12 or m9G4 (mIgG1 isotype control Mab) for 20 min at 4° C. After 3washes in phosphate-buffered saline (PBS) supplemented with 1% BSA and0.01% NaN₃, cells were incubated with secondary antibody goat anti-mouseAlexa 488 (dilution 1/500) for 20 minutes at 4° C. After 3 additionalwashes in PBS supplemented with 1% BSA and 0.1% NaN₃, cells wereanalyzed by FACS (Facscalibur, Becton-Dickinson). At least 5000 cellswere assessed to calculate the mean value of fluorescence intensity.Data are analyzed using Prism software.

Results are represented in FIG. 5. The titration curve established onCOS7 cells using either 1613F12 or mIgG1 isotype control confirms that1613F12 is able to recognize the monkey cellular form of the Axlreceptor expressed on the surface of the COS7 cells. Plateau is reachedfor 1613F12 concentrations above 0.625 μg/ml (4.2 10⁻¹⁰ M). As expectedno binding is observed in presence of the mIgG1 isotype control.

This example illustrates the fact that the 1613F12 does not cross-reactwith the mouse Axl receptor. In contrast it strongly binds to the monkeyAxl receptor expressed on the surface of COS7 cells.

Example 6: Gas6 Competition Experiments Performed in Presence of the1613F12

To further characterize the anti-Axl Mabs, Gas6 competition assays wereperformed. In this assay, the free rhAxl-Fc protein and the anti-Axlantibody are incubated to form antigen-antibody complex and then thecomplexes are loaded on Gas6-coated surface in the assay plate. Theunbound antibody-antigen complexes are washed out before addingenzyme-linked secondary antibody against the human Fc portion of therhAxl-Fc protein. The substrate is then added and the antigenconcentration can be determined by the signal strength elicited by theenzyme-substrate reaction.

Briefly reaction mixture comprising the rhAxl-Fc protein in the presenceor not of the anti-Axl Mabs to be tested, are prepared on a separatesaturated (0.5% gelatin in PBS 1×) plate. Serial 1: 2 dilutions(starting from 80 μg/ml on 12 columns) of murine anti-Axl antibodies areperformed. Then 0.5 μg/ml of the rhAxl-Fc protein is added (R and DSystems, ref 154AL/CF), except to the negative control line thatcontains only ELISA diluant (0.1% gelatin, 0.05% Tween 20 in PBS 1×).After homogenisation, the competition samples are loaded on Gas6-coatedplates with a 6 μg/ml rhGas6 solution in PBS (R and D systems cat No.885-GS-CS/CF). After incubation and several washes, bound rhAxl-Fcproteins are detected using a goat anti-Human IgG-HRP (Jackson, ref109-035-098). Once bound, the TMB substrate is added to the plates. Thereaction is stopped by addition of 1M H₂SO₄ acid solution and theobtained optical densities read at 450 nm using a microplate readerinstrument.

This experiment (FIG. 6) shows that the 1613F12 is able to compete withthe rhAxl-Fc binding on its immobilized ligand. Competition with Gas6binding occurs in presence of 1613F12 antibody concentrations above 2.5μg/ml (1.67 10⁻⁸ M). No more binding of the rhAxl-Fc on the immobilizedGas6 is observed in presence of a 1613F12 concentration above 10 μg/ml(6.67 10⁻⁸ M). The 1613F12 blocks Gas6 binding to rhAxl-Fc.

Example 7: Epitope Recognition by Western Blot

To determine if the 1613F12 recognizes a linear or a conformationalepitope, western blot analysis was done using SN12C cell lysates.Samples were differently treated to be in reducing or non reducingconditions. If a band is visualized with reduced sample, the testedantibody targets a linear epitope of the ECD domain; If not, it israised against a conformation epitope of the Axl ECD.

SN12C cells were seeded in RPMI+10% heat inactivated FBS+2 mML-glutamine at 5.10⁴ cells/cm² in T162 cm² flasks for 72 h at 37° C. ina 5% CO₂ atmosphere. Then the cells were washed twice with phosphatebuffered saline (PBS) and lysed with 1.5 ml of ice-cold lysis buffer [50mM Tris-HCl (pH7.5); 150 mM NaCl; 1% Nonidet P40; 0.5% deoxycholate; and1 complete protease inhibitor cocktail tablet plus 1% antiphosphatases].Cell lysates were shaken for 90 min at 4° C. and cleared at 15 000 rpmfor 10 min. Protein concentration was quantified using BCA. Varioussamples were loaded. First 10 μg of whole cell lysate (10 μg in 20 μl)were prepared in reducing conditions (1× sample buffer (BIORAD)+1×reducing agent (BIORAD)) and loaded on a SDS-PAGE after 2 min incubationat 96° C. Secondly two other samples of 10 μg of whole cell lysate wereprepared in non-reducing conditions (in 1× sample buffer (BIORAD) only).Prior to be loaded on the SDS-PAGE gel, one of these two last samples isheated 2 min incubation at 96° C.; the other one is kept on ice. Aftermigration, the proteins are transferred to nitrocellulose membrane.Membranes were saturated for 1 h at RT with TBS-tween 20 0.1% (TBST), 5%non-fat milk and probed with the 1613F12 at 10 μg/ml overnight at 4° C.Antibodies were diluted in Tris-buffered saline-0.1% tween 20 (v/v)(TBST) with 5% non-fat dry milk. Then membranes were washed with TBSTand incubated with peroxydase-conjugated secondary antibody (dilution1/1000) for 1 h at RT. Immunoreactive proteins were visualized with ECL(Pierce #32209). After Axl visualization, membranes were washed onceagain with TBST and incubated for 1 h at RT with mouse anti-GAPDHantibody (dilution 1/200 000). Then membranes were washed in TBST andincubated with peroxydase-conjugated secondary antibodies, for 1 h atRT. Membranes were washed and GAPDH was revealed using ECL.

Results are represented in FIG. 7.

The 1613F12 mainly recognizes a conformational epitope as a specificband is essentially observed in non-reduced conditions. However a faintsignal is detected in the denaturating migrating condition of the SN12Ccell lysate indicating 1613F12 is able to weakly bind to a linearepitope.

Example 8: Measurement of Axl Down-Regulation Triggered by the 1613F12and Other Anti-Axl Antibodies by Western Blot

In the following example, the human renal cell carcinoma cell line SN12C(ATCC) was selected to address the activity of Axl antibodies on Axlreceptor expression. The SN12C cell line overexpresses the Axl receptor.The Axl down-regulation was studied by Western-Blot on whole cellextracts.

SN12C cells were seeded in RPMI+10% heat inactivated FBS+2 mML-glutamine at 6·10⁴ cells/cm² in six-well plates for 48 h at 37° C. ina 5% CO₂ atmosphere. After two washes with phosphate buffer saline(PBS), cells were serum-starved in a medium containing either 800 ng/mlrecombinant mouse gas6 ligand (R and D Systems, ref: 986-GS/CF) or 10μg/ml of a mIgG1 isotype control antibody (9G4) or 10 μg/ml of the Axlantibody of the present invention and incubated for 4 h or 24 additionalhours. Then the medium was gently removed and cells washed twice withcold PBS. Cells were lysed with 200 μl of ice-cold lysis buffer [50 mMTris-HCl (pH7.5); 150 mM NaCl; 1% Nonidet P40; 0.5% deoxycholate; and 1complete protease inhibitor cocktail tablet plus 1% antiphosphatases].Cell lysates were shaken for 90 min at 4° C. and cleared at 15 000 rpmfor 10 min. Protein concentration was quantified using BCA method. Wholecell lysates (10 μg in 20 μl) were separated by SDS-PAGE and transferredto nitrocellulose membrane. Membranes were saturated for 1 h at RT withTBS-Tween 20 0.1% (TBST), 5% non-fat milk and probed with a commercialM02 Axl antibody at 0.5 μg/ml (AbNova H00000558-M02) overnight at 4° C.Antibodies were diluted in Tris-buffered saline-0.1% tween 20 (v/v)(TBST) with 5% non-fat dry milk. Then membranes were washed with TBSTand incubated with peroxydase-conjugated secondary antibody (dilution1/1000) for 1 h at RT. Immunoreactive proteins were visualized with ECL(Pierce #32209). After Axl visualization, membranes were washed onceagain with TBST and incubated for 1 h at RT with mouse anti-GAPDHantibody (dilution 1/200000). Then membranes were washed in TBST andincubated with peroxydase-conjugated secondary antibodies, for 1 h atRT. Membranes were washed and GAPDH was revealed using ECL. Bandintensity was quantified by densitometry.

Experimental data describing the effect of the isotype control, rmGas6and the 1613F12 antibody are provided in FIGS. 8A-8B. They arerepresentative of 3 independent experiments. Then the percentage ofdownregulated Axl receptor in the SN12C cell extract at 24 h obtained inpresence of other Axl antibodies is given in Table 12.

The results show that 1613F12 is able to down-regulate Axl in anAxl-overexpressing human tumor cell line. At 4 h, the 1613F12 triggers a66% Axl down-regulation, and up to 87% after a 24 hour incubation withthe 1613F12. For the other tested anti-Axl antibodies, the percentage ofdownregulated Axl in SN12C cells after a 24 h incubation of Axl antibodyis comprised between 48.7% and 89%.

TABLE 12 Axl downregulation after Axl antibody binding on SN12C cellsstudied by Western Blot % downregulated Axl at 24 h 1A4 69.0 11D12 64.4320G10 71.0 427B7 62.0 517F1 68.0 530C8 48.7 534G9 64.5 535F7 80.0 547A180.0 1023F10 70.0 1614G5 63.0

Example 9: Flow Cytometry Study of the 1613F12 and Other Anti-AxlAntibodies Effect on Cell Surface Axl Expression

Flow cytometry technique allows labelling of cell-surface Axl receptor.The use of this technique can highlight the effect of antibody on themembrane Axl expression. Human renal tumor SN12C cells that express highlevels of Axl were used in this example.

SN12C tumor cell line was cultured in RMPI1640 with 1% L-glutamine and10% of FCS for 3 days before experiment. Cells were then detached usingtrypsin and plated in 6-multiwell plate in RPMI1640 with 1% L-glutamineand 5% FBS. The next day, antibodies of interest were added at 10 μg/ml.Untreated wells were also included. The cells are incubated at 37° C.,5% CO₂. Twenty four hours later, cells were washed with PBS, detachedand incubated with the same antibodies of interest in FACS buffer (PBS,1% BSA, 0.01% sodium azide). Untreated wells were also stained with thesame antibody in order to compare the signal intensity obtained with thesame Mab on the treated and the non-treated cells. Cells were incubatedfor 20 minutes at 4° C. and washed three times with FACS buffer. AnAlexa 488-labeled goat anti-mouse IgG antibody was incubated for 20minutes and cells were washed three times before FACS analysis onpropidium iodide negative cell population.

Two parameters are determined: (i) the difference of the fluorescentsignal detected on the surface of untreated (no Ab) cells compared tothe Ab-treated cells at T24 h and (ii) the percentage of remaining Axlon the cell surface. The percentage of remaining Axl is calculated asfollows:% remaining Axl=(MFI_(Ab 24 h)/MFI_(no Ab 24 h))×100

Data from one representative experiment (out of 3) are presented inTable 13a. The results were reproduced in three independent experiments.

The difference of MFI between the staining of a Mab in the untreatedcell and the treated condition with the same antibody reflects adown-regulation of the Axl protein on the cell surface of the cells dueto the binding of the considered Mab. Conditions without antibody gavesimilar results to conditions in presence of the isotype controlantibody (m9G4).

TABLE 13a MFI at Δ (MFI_(No Ab 24 h) − Labelling Treatment T24 hMFI_(Ab 24 h)) % remaining Axl 1613F12 No Ab 938 514 45.2 1613F12 4249G4 No Ab 11 −2 117 9G4 13 MAB154 No Ab 950 ND ND 9G4 ND

The data demonstrate that the mean fluorescence intensity detected onthe surface of the cells treated with 1613F12 for 24 hours is reduced(−514) compared to the MFIs obtained with untreated cells labelled withthe 1613F12. After a 24 h incubation with the 1613F12 antibody, 45.2% ofthe cell-surface Axl receptor remains at the SN12C cell-surface.

Data obtained with other anti-Axl antibodies are presented in Table 13b.

TABLE 13b Antibody internalization study by flow cytometry usnig SN12Ccells ΔMFI 1A4 497 11D12 438 320G10 355 427B7 256 517F1 303 530C8 334534G9 257 535F7 352 547A1 473 1023F10 380 1614G5 305

Example 10: Anti-Axl Antibodies Internalization Study Using FluorescentImmunocytochemistry Labelling

Complementary internalization results are obtained by confocalmicroscopy using indirect fluorescent labelling method.

Briefly, SN12C tumor cell line was cultured in RMPI1640 with 1%L-glutamine and 10% of FCS for 3 days before experiment. Cells were thendetached using trypsin and plated in 6-multiwell plate containingcoverslide in RPMI1640 with 1% L-glutamine and 5% FCS. The next day, theanti-Axl antibody was added at 10 μg/ml. Cells treated with anirrelevant antibody were also included. The cells were then incubatedfor 1 h and 2 h at 37° C., 5% CO₂. For T 0 h, cells were incubated for30 minutes at 4° C. to determine antibody binding on cell surface. Cellswere washed with PBS and fixed with paraformaldehyde for 15 minutes.Cells were rinsed and incubated with a goat anti-mouse IgG Alexa 488antibody for 60 minutes at 4° C. to identify remaining antibody on thecell surface. To follow antibody penetration into the cells, cells werefixed and permeabilized with saponin. A goat anti-mouse IgG Alexa 488(Invitrogen) was used to stained both the membrane and the intracellularantibody. Early endosomes were identified using a rabbit polyclonalantibody against EEA1 revealed with a goat anti-rabbit IgG-Alexa 555antibody (Invitrogen). Cells were washed three times and nuclei werestained using Draq5. After staining, cells were mounted in Prolong Goldmounting medium (Invitrogen) and analyzed by using a Zeiss LSM 510confocal microscope.

Photographs are presented in FIGS. 9A-9C.

Images were obtained by confocal microscopy. In presence of the mIgG1isotype control (9G4), neither membrane staining nor intracellularlabelling is observed (FIG. 9A). A progressive loss of the membraneanti-Axl labelling is observed as soon as after 1 h incubation of theSN12C cells with the 1613F12 (FIG. 9B). Intracellular accumulation ofthe 1613F12 Axl antibody is clearly observed at 1 h and 2 h (FIG. 9C).Intracellular antibody co-localizes with EEA1, an early endosome marker.These photographs confirm the internalization of the 1613F12 into SN12Ccells.

Example 11: In Vitro Anti-Axl Mediated Anti-Tumoral Activity

SN12C Proliferation Assay

Ten thousand SN12C cells per well were seeded in FCS-free medium on 96well plates over night at 37° C. in a 5% CO₂ atmosphere. The next day,cells were pre-incubated with 10 μg/ml of each antibody for 1 h at 37°C. Cells were treated with or without rmGas6 (R and D Systems, cat No.986-GS/CF), by adding the ligand directly to the well, and then left togrown for 72 h. Proliferation was measured following ³H thymidineincorporation.

Data are presented in FIG. 10. No effect was observed with the 1613F12which is silent when added to SN12C cells.

Example 12: Cytotoxicity Potency of 1613F12-Saporin and of 1A4-SaporinImmunoconjugates in Various Human Tumor Cell Lines

In the present example, is first documented the cytotoxicity potency ofthe saporin coupled-1613F12. For this purpose direct in vitrocytotoxicity assays using a large panel of human tumor cell lines wereperformed (FIGS. 11A-11K). This tumor cell line panel offers various Axlexpression levels. Secondly, is evaluated another anti-Axl targetingimmunoconjugate, 1A4-saporin for its direct cytotoxic effect on humanSN12C renal tumor cells (FIG. 13).

Briefly, 5000 cells were seeded in 96 well culture plates in 100 μl of5% FBS adequate culture medium. After 24 hour incubation in a 5% CO₂atmosphere at 37° C., a range of concentrations of the anti-Axl antibodyor the immunoconjugate (anti-Axl antibody-saporin or 9G4-saporin or thenaked anti-Axl antibody or 9G4) is applied to the cells. Theimmunoconjugates were used in a large range of concentrations. Cultureplates are then incubated at 37° C. in a humidified 5% CO₂ incubator for72 hours.

At D4, the cell viability is assessed using the CellTiter-Glo®Luminescent Cell Viability kit (Promega Corp., Madison, Wis.) thatallows determining the number of viable cells in culture based onquantification of the ATP present, an indicator of metabolically activecells. Luminescent emissions are recorded by a luminometer device.

From luminescence output is calculated the percentage of cytotoxicityusing the following formula:% cytotoxicity=100−[(RLU _(Ab-sap)×100)/RLU _(No Ab)]

On FIGS. 11A-11K are put together graphs presenting cytotoxicitypercentage in function of the immunoconjugate concentration obtained indistinct in vitro cell cytotoxicity assays with (A) SN12C, (B) Calu-1,(C) A172, (D) A431, (E) DU145, (F) MDA-MB-435S, (G) MDA-MB-231, (H) PC3,(I) NCI-H226, (J) NCI-H125 or (K) Panc1 tumor cells treated with a rangeof 1613F12-saporin immunoconjugate concentrations.

FIGS. 11A-11K shows that the 1613F12-saporin immunoconjugate triggeredcytotoxicity in these different human tumor cell lines. The potency ofthe resulting cytotoxicity effect depends on the human tumor cell line.

FIG. 13 presents the cytotoxicity activity triggered by the 1A4-saporinimmunoconjugate using SN12C human renal cells. This Axl-targetingimmunoconjugate exhibited a dose-dependent cytotoxic effect with amaximum cytotoxic effect of 71%,

Example 13: Binding Kinetics of Axl Antibodies to Human Axl ECD

Affinity measurement of the 1613F12 was then determined using Biacore.

A Biacore X is used to measure the binding kinetics of Axl antibodies onhuman Axl ECD.

The instrument based on the optical phenomenon of surface plasmonresonance (SPR) used by Biacore systems enables the detection andmeasurement of protein-protein interactions in real time, without theuse of labels.

Briefly, the experiments were realized using a sensor chip CM5 as thebiosensor. Rabbit IgGs were immobilized on the flow cells 1 and 2 (FC1and FC2) of a CM5 sensor chip at a level of 9300-10000 response units(RU) using amine coupling chemistry to capture antibodies.

Binding is evaluated using multiple cycles. Each cycle of measure isperformed using a flow rate of 30 μl/min in a HBS-EP buffer. Then theAxl antibody to be tested is captured on the chip for 1 min on FC2 onlyto reach a mean capture value of 311.8 RU (SD=5.1 RU) for the 1613F12antibody. The analyte (Axl ECD antigen) is injected starting at 200 nMand using two-fold serial dilutions to measure rough ka and kd in realtime.

At the end of each cycle, the surfaces are regenerated by injecting a 10mM glycine hydrochloride pH1.5 solution to eliminate theantibody-antigen complexes and the capture antibody as well. Theconsidered signal corresponds to the difference of the signals observedbetween FC1 and FC2 (FC2−FC1). Association rates (ka) and dissociationrates (kd) were calculated using a one-to-one Langmuir binding model.The equilibrium dissociation constant (KD) is determined as the ka/kdratio. The experimental values were analyzed in the Biaevaluationsoftware version 3.0. A χ2 analysis will be performed to assess theaccuracy of the data.

Data are summarized in the following Table 14.

TABLE 14 Binding kinetics and affinity of 1613F12 to human Axl ECDAntibody Ka (1/Ms) Kd (1/s) KD (M) Chi² 1613F12 1.06 10⁵ 2.42 10⁻⁴ 2.2910⁻⁹ 0.71 (0.6%)

To produce the human extracellular domain (ECD) of Axl, the human cDNAscoding for the human soluble AXL receptor was first cloned into thepCEP4 expression vector by PCR. The purified product was then digestedwith restriction enzymes HindIII and BamHI and ligated into pCEP4expression vector which had been precut with the same enzymes. Finally,the identified recombinant plasmid pCEP[AXL]His₆ was further confirmedby DNA sequencing.

Then suspension adapted cells HEK293E were cultivated in Ex-cell 293(SAFC Biosciences) medium with 4 mM glutamine. All transfections wereperformed using linear 25 kDa polyethyleneimine (PEI). The transfectedcultured were maintained at 37° C. in an incubateur shaker with 5% CO2and with agitation at 120 rpm for 6 days. The cells were collected bycentrifugation, and the supernatant containing the recombinantHis-tagged protein was treated for purification on a Ni-NTA agarosecolumn.

The invention claimed is:
 1. An in vitro method for the screening of anantigen-binding protein, or a binding fragment thereof, capable ofdelivering or internalizing a molecule of interest into a mammalian cellexpressing at its surface the Axl protein, said molecule of interestbeing covalently linked to said antigen binding protein, said methodcomprising the steps of: a) selecting an antigen binding protein whichis capable of specifically binding the Axl protein, wherein saidantigen-binding protein has no significant activity on the proliferationof tumor cells; b) covalently linking the molecule of interest to theantigen binding protein selected in step a) to form a complex; c)contacting the complex obtained in step b), with a mammalian cellexpressing at its surface the Axl protein; d) determining whether saidcomplex has been intracellularly delivered or internalized into the saidmammalian cell expressing at its surface the Axl protein; and e)selecting said antigen binding protein as a compound capable ofdelivering or internalizing a molecule of interest into a mammalian cellexpressing at its surface the Axl protein.
 2. The method of claim 1,wherein the antigen binding protein selected in step a) is capable ofbinding the human protein Axl.
 3. The method of claim 1, wherein theantigen binding protein selected in step a) is capable of binding thehuman protein Axl extracellular domain (ECD) with an EC50 of at least10⁻⁹ M.
 4. The method of claim 1, wherein the determination in step d)is performed by a method selected from the group of FACS,Immunofluorescence, flow cytometry, western-blot andcytotoxicity/cytostatic evaluations methods.
 5. An in vitro method forthe preparation of a cytotoxic or cytostatic complex capable ofdelivering a cytotoxic compound into a mammalian cell expressing at itssurface the Axl protein, said method comprising the step of: covalentlylinking a cytotoxic or cytostatic agent to an antigen binding protein,wherein said antigen binding protein is: i) capable of specificallybinding the Axl protein, ii) devoid of any significant activity on theproliferation of tumor cells, and iii) internalized into a mammaliancell following its binding to said Axl protein when said Axl protein isexpressed at the surface of said mammalian cell.
 6. The in vitro methodaccording to claim 5, wherein said antigen binding capable ofspecifically binding the Axl protein and internalized into a mammaliancell following its binding to said protein Axl when said Axl protein isexpressed at the surface of said mammalian cell, is an antigen bindingprotein selected by an in vitro method comprising the steps of: a)selecting an antigen binding protein which is capable of specificallybinding the Axl protein, wherein said antigen-binding protein has nosignificant activity on the proliferation of tumor cells; b) covalentlylinking the molecule of interest to the antigen binding protein selectedin step a) to form a complex; c) contacting the complex obtained in stepb), with a mammalian cell expressing at its surface the Axl protein; d)determining whether said complex has been intracellularly delivered orinternalized into the said mammalian cell expressing at its surface theAxl protein; and e) selecting said antigen binding protein as a compoundcapable of delivering or internalizing a molecule of interest into amammalian cell expressing at its surface the Axl protein.
 7. An in vitromethod for the preparation of a cytotoxic or cytostatic complex capableof delivering a cytotoxic compound into a mammalian cell, said methodcomprising the step of: covalently linking a cytotoxic agent to acompound which is: i) capable of specifically binding the Axl protein,ii) devoid of any significant activity on the proliferation of tumorcells, and iii) is internalized into a mammalian cell following itsbinding to said protein Axl when said Axl protein is expressed at thesurface of said mammalian cell.
 8. The in vitro method according toclaim 7, wherein said compound capable of specifically binding the Axlprotein and internalized into a mammalian cell following its binding tosaid protein Axl when said Axl protein is expressed at the surface ofsaid mammalian cell, is an antigen binding protein selected by an invitro method comprising the steps of: a) selecting an antigen bindingprotein which is capable of specifically binding the Axl protein,wherein said antigen-binding protein has no significant activity on theproliferation of tumor cells; b) covalently linking the molecule ofinterest to the antigen binding protein selected in step a) to form acomplex; c) contacting the complex obtained in step b), with a mammaliancell expressing at its surface the Axl protein; d) determining whethersaid complex has been intracellularly delivered or internalized into thesaid mammalian cell expressing at its surface the Axl protein; and e)selecting said antigen binding protein as a compound capable ofdelivering or internalizing a molecule of interest into a mammalian cellexpressing at its surface the Axl protein.
 9. The method of claim 1,wherein the antigen binding protein selected in step a) is capable ofbinding the extracellular domain (ECD) of the human Axl protein, saidECD having the sequence SEQ ID No. 31 or
 32. 10. The method of claim 1,wherein the antigen binding protein selected in step a) is capable ofbinding the human Axl protein extracellular domain (ECD) with an EC50comprised between 10⁻⁹ and 10⁻¹² M.
 11. The method of claim 5, whereinthe Axl protein of step a) is the human Axl protein.
 12. The method ofclaim 7, wherein the Axl protein of step a) is the human Axl protein.